Method for the production of polyunsaturated fatty acids

ABSTRACT

The present invention relates to an improved process for the specific production of poly-unsaturated omega-3 and omega-6 fatty acids and a process for the production of triglycerides having an increased content of unsaturated fatty acids, in particular omega-3 and omega-6 fatty acids having at least two double bonds and a 20 or 22 carbon atom chain length. The invention relates to the production of a transgenic organism, preferably a transgenic plant or a transgenic microorganism, hav-ing an increased content of fatty acids, oils or lipids containing C20- or C22-fatty acids with a delta-5, 7, 8, 10 double bond, respectively due to the expression of a delta-8-desaturase and a delta-9-elon-gase from organisms such as plants preferably Algae like  Isochrysis galbana  or  Euglena gracilis . In addition the invention relates to a process for the production of poly unsaturated fatty acids such as Eicosapentaenoic, Arachidonic, Docosapentaenoic or Doosahexaenoic acid through the co-expression of a delta-8-desaturase, a delta-9-elongase and a delta-5 desaturase in organisms such as microorganisms or plants. The invention additionally relates to the use of specific nucleic acid sequences encoding for the aforementioned proteins with delta-8-desaturase-, delta-9-elongase- or delta-5-desaturase-activity, nucleic acid constructs, vectors and organisms containing said nucleic acid sequences. The invention further relates to unsaturated fatty acids and triglycerides having an increased content of at least 1% by weight of unsaturated fatty acids and use thereof.

RELATED APPLICATIONS

This application is a national stage application (under 35 U.S.C. 371)of PCT/EP2003/014054 filed Dec. 11, 2003 which claims benefit to GreatBritain application 0229578.0 filed Dec. 19, 2002 and Great Britainapplication 0316989.3 filed Jul. 23, 2003.

FIELD OF THE INVENTION

The present invention relates to an improved process for the specificproduction of poly-unsaturated ω-3 and ω-6 fatty acids and a process forthe production of triglycerides having an increased content ofunsaturated fatty adds, in particular ω-3 and ω-6 fatty acids having atleast two double bonds and a 20 or 22 carbon atom chain length. Theinvention relates to the production of a transgenic organism, preferablya transgenic plant or a transgenic microorganism, having an increasedcontent of fatty acids, oils or lipids containing C₂₀- or C₂₂-fattyacids with a Δ 5, 7, 8, 10 double bond, respectively due to theexpression of a Δ 8-desaturase and a Δ 9-elongase from organisms such asplants preferably Algae like Isochrysis galbana or Euglena gracilis. Inaddition the invention relates to a process for the production of polyunsaturated fatty acids such as Eicosapentaenoic, Arachidonic,Docosapentaenoic or Docosahexaenoic acid through the co-expression of aΔ-8-desaturase, a Δ-9-elongase and a Δ-5 desaturase in organisms such asmicroorganisms or plants.

The invention additionally relates to the use of specific nucleic acidsequences encoding for the aforementioned proteins with Δ-8-desaturase-,Δ-9-elongase- or Δ-5-desaturase-activity, nucleic acid constructs,vectors and organisms containing said nucleic acid sequences. Theinvention further relates to unsaturated fatty adds and triglycerideshaving an increased content of at least 1% by weight of unsaturatedfatty acids and use thereof.

DESCRIPTION OF RELATED ART

Fatty acids and triglycerides have numerous applications in the foodindustry, animal nutrition, cosmetics and in the drug sector. Dependingon whether they are free saturated or unsaturated fatty acids ortriglycerides with an increased content of saturated or unsaturatedfatty acids, they are suitable for the most varied applications; thus,for example, polyunsaturated fatty acids (=PUFAs) are added to infantformula to increase its nutritional value. The various fatty acids andtriglycerides are mainly obtained from microorganisms such asMortierella or from oil-producing plants such as soybean, oilseed rape,sunflower and others, where they are usually obtained in the form oftheir triacylglycerides. Alternatively, they are obtained advantageouslyfrom animals, such as fish. The free fatty acids are preparedadvantageously by hydrolysis.

Whether oils with unsaturated or with saturated fatty acids arepreferred depends on the intended purpose; thus, for example, lipidswith unsaturated fatty acids, specifically polyunsaturated fatty acids,are preferred in human nutrition since they have a positive effect onthe cholesterol level in the blood and thus on the possibility of heartdisease. They are used in a variety of dietetic foodstuffs ormedicaments. In addition PUFAs are commonly used in food, feed and inthe cosmetic industry. Poly unsaturated ω-3- and/or ω-6-fatty acids arean important part of animal feed and human food. Because of the commoncomposition of human food poly unsaturated ω-3-fatty acids, which are anessential component of fish oil, should be added to the food to increasethe nutritional value of the food; thus, for example, poly unsaturatedfatty acids such as Docosahexaenoic acid (=DHA, C_(22:5)^(Δ4,7,10,13,16,19)) or Eicosapentaenoic acid (=EPA, C_(20:5)^(Δ5,8,11,14,17)) are added as mentioned above to infant formula toincrease its nutritional value. Whereas DHA has a positive effect of thebrain development of babies. The addition of poly unsaturated ω-3-fattyacids is preferred as the addition of poly unsaturated ω-6-fatty acidslike Arachidonic acid (=ARA, C_(220:4) ^(Δ5,8,11,14)) to common foodhave an undesired effect for example on rheumatic diseases such asrheumatoid arthritis. Poly unsaturated ω-3- and ω-6-fatty acids areprecursor of a family of paracrine hormones called eicosanoids such asprostaglandins which are products of the metabolism of Dihomo-γ-linoleicacid, ARA or EPA. Eicosanoids are involved in the regulation oflipolysis, the initiation of inflammatory responses, the regulation ofblood circulation and pressure and other central functions of the body.Eicosanoids comprise prostaglandins, leukotrienes, thromboxanes, andprostacyclins. ω-3-fatty acids seem to prevent artherosclerosis andcardiovascular diseases primarily by regulating the levels of differenteicosanoids. Other Eicosanoids are the thromboxanes and leukotrieneswhich are products of the metabolism of ARA or EPA.

Principally microorganisms such as Mortierella or oil producing plantssuch as soybean, rapeseed or sunflower or algae such as Crytocodinium orPhaeodactylum are a common source for oils containing PUFAs, where theyare usually obtained in the form of their triacyl glycerides.Alternatively, they are obtained advantageously from animals, such asfish. The free fatty acids are prepared advantageously by hydrolysiswith a strong base such as potassium or sodium hydroxide. Higher polyunsaturated fatty acids such as DHA, EPA, ARA, Dihomo-γ-linoleic acid(C_(20:3) ⁶⁶ 8,11,14) or Docosapentaenoic acid (=DPA, C_(22:5)^(Δ7,10,13,16,19)) are not produced by oil producing plants such assoybean, rapeseed, safflower or sunflower. A natural sources for saidfatty acids are fish for example herring, salmon, sardine, redfish, eel,carp, trout, halibut, mackerel, pikeperch or tuna or algae.

On account of their positive properties there has been no shortage ofattempts in the past to make available genes which participate in thesynthesis of fatty acids or triglycerides for the production of oils invarious organisms having a modified content of unsaturated fatty acids.Thus, in WO 91/13972 and its US equivalent a Δ-9-desaturase isdescribed. In WO 93/11245 a Δ-15-desaturase and in WO 94/11516 aΔ-12-desaturase is claimed. WO 00/34439 discloses a Δ-5- and aΔ-8-desaturase. Other desaturases are described, for example, in EP-A-0550 162, WO 94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0 794250, Stukey et al., J. Biol. Chem., 265, 1990: 20144-20149, Wada et al.,Nature 347, 1990: 200-203 or Huang et al., Lipids 34, 1999: 649-659. Todate, however, the various desaturases have been only inadequatelycharacterized biochemically since the enzymes in the form ofmembrane-bound proteins are isolable and characterizable only with verygreat difficulty (McKeon et al., Methods in Enzymol. 71,1981:12141-12147, Wang et al., Plant Physiol. Biochem., 26,1988: 777-792).Generally, membrane-bound desaturases are characterized by introductioninto a suitable organism which is then investigated for enzyme activityby means of analysis of starting materials and products. Δ-6-Desaturasesare described in WO 93/06712, U.S. Pat. No. 5,614,393, U.S. Pat. No.5,614,393, WO 96/21022, WO0021557 and WO 99/27111 and their applicationto production in transgenic organisms is also described, e.g. in WO9846763, WO 9846764 and WO 9846765. At the same time the expression ofvarious fatty acid biosynthesis genes, as in WO 9964616 or WO 9846776,and the formation of poly-unsaturated fatty acids is also described andclaimed. With regard to the effectiveness of the expression ofdesaturases and their effect on the formation of polyunsaturated fattyacids it may be noted that through expression of a desaturases andelongases as described to date only low contents of poly-unsaturatedfatty acids/lipids, such as by way of example eicosapentaenoic orarachidonic acid, have been achieved. Therefore, an alternative and moreeffective pathway with higher product yield is desirable.

Accordingly, there is still a great demand for new and more suitablegenes which encode enzymes which participate in the biosynthesis ofunsaturated fatty acids and make it possible to produce certain fattyacids specifically on an industrial scale without unwanted byproductsforming. In the selection of genes for biosynthesis two characteristicsabove all are particularly important. On the one hand, there is as evera need for improved processes for obtaining the highest possiblecontents of polyunsaturated fatty acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the fatty acid profile (FAMes) of leaf tissue from wildtype Arabidopsis thaliana as a control.

FIG. 2 shows the fatty acid profile (FAMes) of leaf tissue fromtrausgenic Arabidopsis expressing the Isochrysis Δ-9-elongase (seeexample 4).

FIG. 3 shows the fatty acid profile (FAMes) of the double transformedArabidopsis line expressing the Isochrysis Δ-9-elongase and the EuglenaΔ-8-desaturase (Line IsoElo X Eu D8 des).

FIG. 4 shows the fatty acid profile (FAMes) of the triple transformedArabidopsis line expressing the Isocbrysis Δ-9-elongase, the EuglenaΔ-8-desaturase, and the Mortierella Δ5 desaturase (Mort Δ5) gene (LineIsoElo X EU D8 des x Mort Δ5).

FIG. 5 shows GC profiles of Arabidopsis leaf fatty acid methyl estersextracted from wild type (FIG. 5A), single transgenic plants expressingIsochrysis galbana Δ9 elongase gene Ig ASE1 (FIG. 5B), double transgenicplant expressing the Ig ASE1 and Euglena Δ8 desaturase (EU Δ8) genes(FIG. 5C), and the triple transfenic plant expressing the Ig ASE1, Eu Δ8and the Mortierella Δ5 desaturase (Mort Δ5) genes (FIG. 5D).

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, it is an object of the present invention to provide furthergenes of desaturase and elongase enzymes for the synthesis ofpolyunsaturated fatty acids in organisms preferably in microorganismsand plants and to use them in a commercial process for the production ofpoly unsaturated fatty acids. Said process should increase PUFA contentin organisms as much as possible preferably in seeds of an oil producingplant.

We have found that this object is achieved by a process for theproduction of compounds of the following general formula

in transgenic organisms with a content of at least 1% by weight of saidcompounds referred to the total lipid content of said organism whichcomprises the following steps:

-   a) introduction of at least one nucleic acid sequence in a    transgenic organism, which encodes a Δ-9-elongase, and-   b) introduction of at least one second nucleic acid sequence which    encodes a Δ-8-desaturase, and-   c) if necessary introduction of at least a one third nucleic acid    sequence, which encodes a Δ-5-desaturase, and-   d) cultivating and harvesting of said organism; and    where the variables and substituents in formula I have the following    meanings:-   R¹=hydroxyl-, Coenzyme A-(Thioester), phosphatidylcholine-,    phosphatidylethanol-amine-, phosphatidylglycerol-,    diphosphatidylglycerol-, phosphabdyiserine-, phosphatidylinositol-,    sphingoflipid-, glycoshingolipid- or a residue of the general    formula II:

where the substituents in formula II have the following meanings:

-   R²=hydrogen-, phosphatidylcholine-, phosphatidylethanolamine-,    phosphatidyglycerol-, diphosphatidylglycerol-, phosphahdyiserine-,    phosphatidylinositol-, shingolipid-, glycoshingolipid-,    glycoshingolipid- or saturated or unsaturated C₂-C₂₄-alkylcarbonyl-,-   R³ =hydrogen-, saturated or unsaturated C₂-C₂₄-alkylcarbonyl-, or-   R² and R³ independent of each other a residue of the formula Ia:

-   n=3,4 or 6, m=3, 4 or 5 and p=0 or 3, preferably n=3, m=4 or 5 and    p=0 or 3.-   R¹ indicates in the formula I hydroxyl-, Acetyl-Coenzyme A-,    phosphatidylcholine-, phosphatidylethanolamine-,    phosphatidylglycerol-, diphosphatidylglyoerol-, phosphatidylserine-,    phosphatidyrinositol-, sphingolipid-, glycoshingolipid- or a residue    of the general formula II

The abovementioned residues for R¹ are always coupled to compounds ofthe general formula I in the form of their ester or thioester.

R² indicates in structures of the general formula II hydrogen,phosphatidylcholine-, phosphatidylethanolamine-, phosphatidylglycerol-,diphosphatidylglycerol-, phosphatidylserine-, phosphatidylinositol-,shingolipid-, glycoshingolipid-, glycoshingolipid- or saturated orunsaturated C₂-C₂₄-alkylcarbonyl-residues,

Alkyl radicals which may be mentioned are substituted or unsubstituted,saturated or unsaturated C₂-C₂₄-alkylcarbonyl-chains such asethylcarbonyl-, n-propylcarbonyl-, n-butylcarbonyl-, n-pentylcarbonyl-,n-hexylcarbonyl-, n-heptylcarbonyl-, n-octylcarbonyl-, n-nonylcarbonyl-,n-decylcarbonyl-, n-undecylcarbonyl-, n-dodecylcarbonyl-,n-tridecylcarbonyl-, n-tetradecylcarbonyl-, n-pentadecylcarbonyl-,n-hexadecylcarbonyl-, n-heptadecylcarbonyl-, n-octadecylcarbonyl-,n-nonadecylcarbonyl-, n-eicosylcarbonyl-, n-docosanylcarbonyl- orn-tetracosanylcarbonyl-, that contain one or more double bonds.Saturated or unsaturated C₁₀-C₂₂-Alkylcarbonylresidues such asn-decylcarbonyl-, n-undecylcarbonyl-, n-dodecylcarbonyl-,n-tridecylcarbonyl-, n-tetradecylcarbonyl-, n-pentadecylcarbonyl-,n-hexadecylcarbonyl-, n-heptadecylcarbonyl-, n-octadecylcarbonyl-,n-nonadecylcarbonyl-, n-eicosylcarbonyl-, n-docosanylcarbonyl- orn-tetracosanylcarbonyl-are preferred, which contain one ore more doublebonds. In particular privileged are saturated or unsaturatedC₁₀-C₂₂-alkylcarbonyl-residue as C₁₀-alkylcarbonyl-, C₁₁-alkylcarbonyl-,C₁₂-alkylcarbonyl-, C₁₃-alkylcarbonyl-, C₁₄-alkylcarbonyl-,C₁₆-alkylcarbonyl-, C₁₈-alkylcarbonyl-, C₂₀-alkylcarbonyl-,C₂₂-alkylcarbonyl- or C₂₄-alkylcarbonyl-residue, that contain one oremore double bonds. In particular privileged are saturated or unsaturatedC₁₆-C₂₂-alkylcarbonyl-residue as C₁₆-alkylcarbonyl-, C₁₈-alkylcarbonyl-,C₂₀-alkylcarbonyl- or C₂₂-alkylcarbonyl-residue, that contain one oremore double bonds. The residues contain in particular two, three, fouror five double bonds. Particularly preferred are residues of 20 or 22carbon atoms having up to five double bonds, preferably three, four orfive double bonds. All residues are derived from the mentionedcorresponding fatty acids.

R³ indicates in structures of the general formula II hydrogen, saturatedor unsaturated C₂-C₂₄-alkylcarbonyl.

Substituted or unsubstituted, saturated or unsaturatedC₂-C₂₄-alkylcarbonyl-residues are e.g. ethylcarbonyl-,n-propylcarbonyl-, n-butylcarbonyl-, n-pentylcarbonyl-,n-hexylcarbonyl-, n-heptylcarbonyl-, n-octylcarbonyl-, n-nonylcarbonyl-,n-decylcarbonyl-, n-undecylcarbonyl-, n-dodecylcarbonyl-,n-tridecylcarbonyl-, n-tetradecylcarbonyl-, n-pentadecylcarbonyl-,n-hexadecylcarbonyl-, n-heptadecylcarbonyl-, n-octadecylcarbonyl-,n-nonadecylcarbonyl-, n-eicosylcarbonyl-, n-docosanylcarbonyl- orn-tetracosanylcarbonyl-, having one or more double bonds. Preferred aresaturated or unsaturated C₁₀-C₂₄-alkylcarbonyl residues asn-decylcarbonyl-, n-undecylcarbonyl-, n-dodecylcarbonyl-,n-tridecylcarbonyl-, n-tetradecylcarbonyl-, n-pentadecylcarbonyl yl-,n-hexadecylcarbonyl-, n-heptadecylcarbonyl-, n-octadecylcarbonyl-,n-nonadecylcarbonyl-, n-eicosylcarbonyl-, n-docosanylcarbonyl- orn-tetracosanylcarbonyl-, with one ore more double bonds. In particularsaturated or unsaturated C₁₀-C₂₄-alkylcarbonyl residues asC₁₀-alkylcarbonyl-, C₁₁-alkylcarbonyl-, C₁₂-alkylcarbonyl-,C₁₃-alkylcarbonyl-, C₁₄-alkylcarbonyl, C₁₆-alkylcarbonyl-,C₁₈-alkylcarbonyl-, C₂₀-alkylcarbonyl-, C₂₂-alkylcarbonyl- orC₂₄-alkylcarbonyl-residues with one or more double bonds. In particularpreferred are saturated or unsaturated C₁₆-C₂₂-alkylcarbonyl-residue asC₁₆-alkylcarbonyl-, C₁₈-alkylcarbonyl-, C₂₀-alkylcarbonyl- orC₂₂-alkylcarbonyl-residues, with multiple double bonds.C₁₈-alkylcarbonyl-residues are particularly preferred, which containone, two, three or four double bonds and C₂₀-alkylcarbonyl-residues,with three, four or five double bonds. All residues are derived from thecorresponding fatty acids.

R² and R³ indicates in structures of the general formula II independentof each other a residue of the general formula Ia

whereas the variables in the formula I and Ia are defined as: n=3,4 or6, m=3, 4 or 5 and p=0 or 3. In particular n=3, m=4 or 5 and p=0 or 3.

The abovementioned residues R¹, R² and R³ can be substituted withhydroxyl- or epoxy-groups or might contain also triple bonds.

According to the invention the used nucleic acid sequences are isolatednucleic sequences coding for polypeptides having C₂₀-Δ5- or Δ-8desaturase or C₁₈-Δ9-elongase activity.

The according to inventive process synthesized substances of formula Iwhich contain as residue R¹ the residue of formula II containpreferentially a mixture of different residues R² or R³. The residuesare derived from different fatty acid molecules as short chain fattyacids with 4 to 6 C-atoms, mid-chain fatty acids having 8 to 12 C-atomsand long-chain fatty acids with 14 to 24 C-atoms, whereas the long-chainfatty acids are preferred. Said long chain fatty acids are derivedpreferentially from C₁₈- or C₂₀-poly unsaturated fatty acids havingadvantageously between two and five double bonds. In addition thebackbone of formula I is also derived from such a aforementioned fattyacid which advantageously is also different from R² and R³. That meanscompounds which are produced by the inventive process are in one aspectof the invention triglycerides of different substituted orunsubstituted, saturated or unsaturated fatty acid ester or thioesters.

According to another aspect of the invention poly-unsaturated fatty acidesters (of the formula I) with 18, 20 or 22 fatty acid carbon atomschain length with at least two double bonds, preferably three, four orfive are particularly preferred.

In particular fatty add molecules with three, four or five double bondsare preferred for the synthesis of eicosadienoic, eicosatrienoic,eicosatetranoic (arachidonic-acid) and eicosapentanoic acid (C20:2n-6,Δ11, 14; C20:3n-6, Δ8, 11, 14; C20:4n-6, Δ5, 8, 11, 14, C20:3n-3, Δ11,14, 17; C20:4n-3, Δ8, 11, 14, 17; C20:5n-3, Δ5, 8, 11, 14, 17) in theinventive process, whereas arachidonic add and eicosapentaenoic acid aremost preferred. We have found that this object is advantageouslyachieved by the combined expression of three isolated nucleic acidsequences according to the invention which encode for polypeptideshaving the following activities: a polypeptides with C20-Δ-8-desaturaseactivity, a C18-Δ-9-elongase activity, and a C20-Δ-5 desaturaseactivity. This objective was achieved in particular by the co-expressionof the isolated nucleic acid sequences according to the invention. C18fatty acids with a double bond in Δ-9-position are elongated by theΔ-9-elongase advantageously used in the inventive process. By theΔ-8-desaturase used in the process a double in Δ-8-position isintroduced into C20 fatty acids. In addition a double bond can beintroduced into the fatty acid molecules in Δ-5-position by theΔ-5-desaturase.

The fatty acid ester of C₁₈-, C₂₀- and/or C₂₂-poly unsaturated fattyacids synthesized in the inventive process advantageously in form oftheir triglycerides as ester or thioesters can be isolated from theproducing organism for example from a microorganism or a plant in theform of an oil, lipid or lipid mixture for example as sphingolipids,phosphoglycerides, lipids, glycolipids such as glycosphingolipids,phospholipids such as phosphatidylethanolamine, phosphatidylcholine,phosphatidylserine, phosphatidylglycerol, phosphatidylinositol ordiphosphatidylglycerol, or as monoacylglyceride, diacylglyceride ortriacylglyceride or as other fatty acid esters such as acetyl-Coenzyme Athioester, which contain saturated or unsaturated fatty acids preferablypoly unsaturated fatty acids with at least two preferably at least threedouble bonds in the fatty acid molecule. In addition to the in form ofthe aforementioned esters bound fatty acids also fatty acids bound inother compounds can be produced or also free fatty acids can be producedby the inventive process.

In general the transgenic organisms for example transgenicmicroorganisms or plants used in the inventive process contain fattyacid esters or fatty acids in a distribution of nearly 80 to 90% byweight of triacyl glycerides, 2 to 5% by weight diacyl glycerides, 5 to10% by weight monoacyl glycerides, 1 to 5% by weight free fatty acidsand 2 to 8% by weight phospholipids, whereas the total amount of theaforementioned compounds are all together a 100% by weight.

In the inventive process(es) [the singular shall include the plural andvice versa] at least 1% by weight, preferably at least 2, 3, 4 or 5% byweight, more preferably at least 6, 7, 8, or 9% by weight, mostpreferably 10, 20 or 30% by weight of the compounds of formula Ireferred to the total lipid content of the organism used in the processare produced. Preferred starting material for the inventive process arelinoleic acid (C18:2) and/or linolenic acid (C18:3) which aretransformed to the preferred end products ARA or EPA. As for theinventive process organisms are used the product of the process is not aproduct of one pure substance per se. It is a mixture of differentsubstances of formula I where one or more compounds are the majorproduct and others are only contained as side products. In the eventthat in an organism used in the process linoleic and linolenic acid areavailable the end product is a mixture of ARA and EPA. Advantageouslythe side products shall not exceed 20% by weight referred to the totallipid content of the organism, preferably the side products shall notexceed 15% by weight, more preferably they shall not exceed 10% byweight, most preferably they shall not exceed 5% by weight. Preferablyorganisms are used in the process which contain as starting materialeither linoleic or linolenic acid so that as end product of the processonly ARA or EPA are produced. In the event EPA and ARA are producedtogether, they should be produced in a ratio of at least 1:2 (EPA-ARA),preferably of at least 1:3, more preferably of at least 1:4, mostpreferably of at least 1:5. In the event that a mixture of differentfatty acids such as ARA and EPA are the product of the inventive processsaid fatty acids can be further purified by method known by a personskilled in the art such as distillation, extraction, crystallization atlow temperatures, chromatography or a combination of said methods.

Advantageously the invented method comprise the following steps:

-   a) expression of at least one nucleic acid sequence in a plant that    codes for an enzyme having Δ-9 elongase activity, and-   b) expression of at least one nucleic acid sequences which codes for    a C20-specific Δ-8 desaturase, and-   c) possibly the expression of a third nucleic acid sequence which    codes for a C20-specific Δ-5 desaturase-   d) followed by the cultivation of the transgenic plants and seed    harvest.

In principle all host organisms can be used in the inventive process forexample transgenic organisms such as plants like mosses; green, red,brown or blue algae; monocotyledons or dicotyledones. Advantageously oilproducing transgenic organisms such as fungi, bacteria, algae, mosses orplants are used in the inventive processes described herein (for theinvention the singular shall include the plural and vice versa),Additional advantageously organisms are animals or preferably plants orparts thereof. Fungi, yeasts or plants are preferably used, particularlypreferably fungi or plants, very particularly preferably plants such asoilseed plants containing high amounts of lipid compounds such asrapeseed, poppy, mustard, hemp, castor bean, sesame, olive, calendula,punica, hazel nut, almond, macadamia, avocado, pumpkin, walnut, laurel,pistachio, primrose, canola, peanut, linseed, soybean, safflower,sunflower, borage or plants such as maize, wheat, rye, oat, triticale,rice, barley, cotton, manihot, pepper, tagetes, solanaceaous plants suchas potato, tobacco, eggplant, and tomato, Vicia species, pea, alfalfa,bushy plants (coffee, cacao, tea), Salix species, trees (oil palm,coconut) and perennial grasses and forage crops. Particularly preferredplants of the invention are oilseed plants rapeseed, poppy, mustard,hemp, castor bean, sesame, olive, calendula, punica, hazel nut, almond,macadamia, avocado, pumpkin, laurel, pistachio, primrose, canola,peanut, linseed, soybean, safflower, sunflower, borage or trees (oilpalm, coconut). Most preferred are C₁₈₋₂- and/or C_(18:3)-fatty acidrich plants such as hemp, sesame, linseed, poppy, pumpkin, walnut,tobacco, cotton, safflower or sunflower.

Depending on the nucleic acid and/or the organism used in the inventiveprocesses different compounds of the general formula I can besynthesized. In addition depending on the plant or fungi used in theprocess different mixtures of formula I compounds or single compoundssuch as arachidonic acid or eicosapentaenoic acid in free or bound formcan be produced. In the event that in the inventive processes organismare used which have as precursor of the fatty acid synthesis preferablyC_(18:2)- or C_(18:3)-fatty acids different poly unsaturated fatty acidscan be synthesized for example starting from C_(18:2)-fatty acidsγ-linoleic acid, dihomo-γ-linoleic acid or arachidonic acid can beproduced or starting from C_(18:3)-fatty acids stearidonic acid,eicosatetraenoic acid or eicosapentaenoic acid can be produced. Byinfluencing the activity of the different genes or their gene productsdifferent single compounds or compound mixtures can be produced. Asliving organisms are used in the inventive process the crude materialthat means crude lipids and/or oils isolated from the organismspreferably contain at least some starting compounds such as C_(18:2)- orC_(18:3)-fatty acids or their combination in the product and dependingon the activity of the nucleic acid sequences and their gene productsfatty acid intermediates of the biosynthesis chain. Said startingcompounds or intermediates are in the product in a concentration of lessthan 20 or 15% by weight, preferably less than 10, 9, 8, 7 or 6% byweight, more preferably less than 5, 4, 3, 2 or 1% by weight of thetotal fatty acids isolated from the used organism.

Transgenic plants are to be understood as meaning single plant cells andtheir cultures on solid media or in liquid culture, parts of plants andentire plants such as plant cell cultures, protoplasts from plants,callus cultures or plant tissues such as leafs, shoots, seeds, flowers,roots etc. Said transgenic plants can be cultivated for example on solidor liquid culture medium, in soil or in hydroponics.

After cultivation transgenic organisms preferably transgenic plantswhich are used in the inventive process can be brought to the marketwithout isolating compounds of the general formula I. Preferably thecompounds of the general formula I are isolated from the organisms inthe form of their free fatty acids, their lipids or oils. Thepurification can be done by conventional methods such as squeezing andextraction of the plants or other methods instead of the extraction suchas distillation, crystallization at low temperatures, chromatography ora combination of said methods. Advantageously the plants are grinded,heated and/or vaporized before the squeezing and extraction procedure.As solvent for the extraction solvents such as hexane are used. Theisolated oils are further purified by acidification with for examplephosphoric acid. The free fatty acids are produced from said oils orlipids by hydrolysis. Charcoal or diatom earth are used to remove dyesfrom the fluid. In another preferred embodiment of the inventive processthe alkyl ester of the fatty acids are produced from the oils and lipidsby transesterification with an enzyme of with conventional chemistry. Apreferred method is the production of the alkyl ester in the presence ofalcohalates of the corresponding lower alcohols (C1 to C10 alcohols suchas methanol, ethanol, propanol, butanol, hexanol etc.) such asmethanolate or ethanolate. Therefore as the skilled worker knows thealcohol in the presence of a catalytic amount of a base such as NaOH orKOH is added to the oils or lipids.

In a preferred form of the inventive process the lipids can be obtainedin the usual manner after the organisms have been grown. To this end,the organisms can first be harvested and then disrupted, or they can beused directly. It is advantageous to extract the lipids with suitablesolvents such as a polar solvents, for example hexane, or polarsolvents, for example ethanol, isopropanol, or mixtures such ashexane/isopropanol, phenol/chloroform/isoamyl alcohol, at temperaturesbetween 0° C. and 80° C., preferably between 20° C. and 50° C. As arule, the biomass is extracted with an excess of solvent, for examplewith an excess of solvent to biomass of 1:4. The solvent is subsequentlyremoved, for example by distillation. The extraction may also be carriedout with supercritical CO₂. After the extraction, the remainder of thebiomass can be removed, for example, by filtration. Standard methods forthe extraction of fatty acids from plants and microorganisms aredescribed in Bligh et al. (Can. J. Biochem. Physiol. 37, 1959: 911-917)or Vick et al. (Plant Physiol. 69, 1982: 1103-1108).

The crude oil thus obtained can then be purified further, for example byremoving cloudiness by adding polar solvents such as acetone or a polarsolvents such as chloroform, followed by filtration or centrifugation.Further purification via columns or other techniques is also possible.

To obtain the free fatty acids from the triglycerides, the latter arehyrolyzed in the customary manner, for example using NaOH or KOH.

In the inventive process oils, lipids and/or free fatty acids orfractions thereof are produced. Said products can be used for theproduction of feed and food products, cosmetics or pharmaceuticals.

In principle all nucleic acids encoding polypeptides withΔ-8-desaturase, Δ-9-elongase and/or Δ-5-desaturase activity can be usedin the inventive process. Preferably the nucleic acid sequences can beisolated for example from microorganism or plants such as fungi likeMortierelia, algae like Euglena, Crypthecodinium or Isochrysis, diatomslike Phaeodactylum or mosses like Physcomla or Ceratodon, but alsonon-human animals such as Caenorhabditis are possible as source for thenuoleic acid sequences. Advantageous nucleic acid sequences according tothe invention which encode polypeptides having a Δ-8-desaturase,Δ-9-elongase and/or Δ-5-desaturase activity are originate frommicroorganisms or plants, advantageously Phaeodactylum tricomutum,Ceratodon purpureus, Physcomitrella patens, Euglena gracilis orIsochrysis galbana. Euglena gracilis or Isochrysis galbana are specificfor the conversion of ω-3- or ω-6 fatty acids. Thus, the co expressionof a Δ-9 elongase and a C20-specific Δ-8-desaturase leads to theformation of eicosatrienoic acid (C20:6n-3, Δ8, 11, 14) andeicosatetraenoic acid (C20:3n-4, Δ8, 11, 14, 17). Co-expression of athird gene coding for a C20-Δ5 specific desaturase leads to theproduction of Arachidonic acid (C20:6n-4, Δ5, 8, 11, 14) orEicosapentaenoic acid (C20:3n-5, Δ5, 8, 11, 14, 17).

By derivative(s) of the sequences according to the invention is meant,for example, functional homologs of the polypeptides or enzymes encodedby SEQ ID NO: 2 or SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ IDNO: 10 which exhibit the same said specific enzymatic activity. Thisspecific enzymatic activity allows advantageously the synthesis ofunsaturated fatty acids having more than three double bonds in the fattyacid molecule. By unsaturated fatty acids is meant in what followsdiunsaturated or polyunsaturated fatty acids which possess double bonds.The double bonds may be conjugated or non conjugated. The said sequencesencode enzymes which exhibit Δ-9 elongase, Δ-8-desaturase or-Δ5-desaturase activity.

The enzyme according to the invention, Δ-9-elongase, Δ-8-desaturase orΔ-5-desaturase, advantageously either elongates fatty acid chains with18 carbon atoms (see SEQ ID NO: 2) or introduces a double bond intofatty acid residues of glycerolipids, free fatty acids or acyl-CoA fattyacids at position C₈-C₉ (see SEQ ID NO: 4) or at position C₅-C₆ (see SEQID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10).

The nucleic acid sequence(s) according to the invention (for purposes ofthe application the singular encompasses the plural and vice versa) orfragments thereof may advantageously be used for isolating other genomicsequences via homology screening.

The said derivatives may be isolated, for example, from other organisms,eukaryotic organisms such as plants, especially mosses, algae,dinoflagellates or fungi, preferably algae and mosses.

Allele variants include in particular functional variants obtainable bydeletion, insertion or substitution of nucleotides in the sequencesdepicted in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 orSEQ ID NO: 9 the enzymatic activity of the derived synthesized proteinsbeing retained.

Starting from the DNA sequence described in SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 or parts of said sequencessuch DNA sequences can be isolated using, for example, normalhybridization methods or the PCR technique from other eukaryotes such asthose identified above for example. These DNA sequences hybridize understandard conditions with the said sequences. For hybridization use isadvantageously made of short oligonucleotides of the conserved regionsof an average length of about 15 to 70 bp, preferably of about 17 to 60bp, more preferably of about 19 to 50 bp, most preferably of about 20 to40 bp, for example, which can be determined by comparisons with otherdesaturase or elongase genes in the manner known to those skilled in theart. The histidine box sequences are advantageously employed. However,longer fragments of the nucleic acids according to the invention or thecomplete sequences may also be used for hybridization. Depending on thenucleic acid employed: oligonucleotide, longer fragment or completesequence, or depending on which type of nucleic acid, DNA or RNA, isused for hybridization these standard conditions vary. Thus, forexample, the melting temperatures of DNA:DNA hybrids are approximately10° C. lower than those of DNA:RNA hybrids of the same length.

By standard conditions is meant, for example, depending on the nucleicacid in question temperatures between 42° C. and 58° C. in an aqueousbuffer solution having a concentration of between 0.1 and 5×SSC(1×SSC=0.15 M NaCl, 15 mM sodium citrate, pH 7.2) or additionally in thepresence of 50% fornamide, such as by way of example 42° C. in 5×SSC,50% formamide. Hybridization conditions for DNA:DNA hybrids areadvantageously 0.1×SSC and temperatures between approximately 20° C. and45° C., preferably between approximately 30° C. and 45° C. For DNA:RNAhybrids the hybridization conditions are advantageously 0.1×SSC andtemperatures between approximately 30° C. and 55° C., preferably betweenapproximately 45° C. and 55° C. These specified temperatures forhybridization are melting temperature values calculated by way ofexample for a nucleic acid having a length of approximately 100nucleotides and a G+C content of 50% in the absence of formamide. Theexperimental conditions for DNA hybridization are described in relevantgenetics textbooks such as by way of example Sambrook et al., “MolecularCloning”, Cold Spring Harbor Laboratory, 1989, and may be calculated byformulae known to those skilled in the art, for example as a function ofthe length of the nucleic acids, the nature of the hybrids or the G+Ccontent. Those skilled in the art may draw on the following textbooksfor further information on hybridization: Ausubel et al. (eds), 1985,Current Protocols in Molecular Biology, John Wiley & Sons, New York;Hames and Higgins (eds), 1985, Nucleic Acids Hybridization: A PracticalApproach, IRL Press at Oxford University Press, Oxford; Brown (ed),1991, Essential Molecular Biology, A Practical Approach, IRL Press atOxford University Press, Oxford.

Furthermore, by derivatives is meant homologs of the sequences SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, forexample eukaryotic homologs, truncated sequences, single-stranded DNA ofthe encoding and nonencoding DNA sequence or RNA of the encoding andnonencoding DNA sequence.

In addition, by homologs of the sequences SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9 is meant derivatives such asby way of example promoter variants. These variants may be modified byone or more nucleotide exchanges, by insertion(s) and/or deletion(s)without, however, adversely affecting the functionality or efficiency ofthe promoters. Furthermore, the promoters can have their efficiencyincreased by altering their sequence or be completely replaced by moreeffective promoters even of foreign organisms.

By derivatives is also advantageously meant variants whose nucleotidesequence has been altered in the region from −1 to −2000 ahead of thestart codon in such a way that the gene expression and/or the proteinexpression is modified, preferably increased. Furthermore, byderivatives is also meant variants which have been modified at the 3′end.

The nucleic acid sequences according to the invention which encode aΔ-8-desaturase, a Δ-5-desaturase and/or a Δ-9-elongase may be producedby synthesis or obtained naturally or contain a mixture of synthetic andnatural DNA components as well as consist of various heterologousΔ-8-desaturase, Δ-5-desaturase and/or Δ-9-elongase gene segments fromdifferent organisms. In general, synthetic nucleotide sequences areproduced with codons which are preferred by the corresponding hostorganisms, plants for example. This usually results in optimumexpression of the heterologous gene. These codons preferred by plantsmay be determined from codons having the highest protein frequency whichare expressed in most of the plant species of interest. An exampleconcerning Corynebacterium glutamicum is provided in Wada et al. (1992)Nucleic Acids Res. 20:2111-2118). Such experiments can be carried outusing standard methods and are known to the person skilled in the art.

Functionally equivalent sequences which encode the Δ-8-desaturase,Δ-5-desaturase and/or Δ-9-elongase gene are those derivatives of thesequence according to the invention which despite differing nucleotidesequence still possess the desired functions, that is to say theenzymatic activity and specific selectivity of the proteins. Thus,functional equivalents include naturally occurring variants of thesequences described herein as well as artificial ones, e.g. artificialnucleotide sequences adapted to the codon use of a plant which have beenobtained by chemical synthesis.

In addition, artificial DNA sequences are suitable, provided, asdescribed above, they mediate the desired property, for example anincrease in the content of Δ-8 and/or Δ-5 double bonds in fatty acids,oils or lipids in organisms such as in a plant by over-expression of theΔ-8- and/or Δ-5-desaturase gene in preferably in crop plants. Suchartificial DNA sequences can exhibit Δ-8 and/or Δ-5-desaturase and/orΔ-9-elongase activity, for example by back-translation of proteinsconstructed by means of molecular modeling, or be determined by in vitroselection. Possible techniques for in vitro evolution of DNA to modifyor improve the DNA sequences are described in Patten, P. A. et al.,Current Opinion in Biotechnology 8, 724-733(1997) or in Moore, J. C. etal., Journal of Molecular Biology 272, 336-347 (1997). Particularlysuitable are encoding DNA sequences which are obtained byback-translation of a polypeptide sequence in accordance with the codonuse specific to the host plant. Those skilled in the art familiar withthe methods of plant genetics can easily determine the specific codonuse by computer analyses of other known genes of the plant to betransformed.

Other suitable equivalent nucleic acid sequences which may be mentionedare sequences that encode fusion proteins, a component of the fusionprotein being a Δ-8- and/or a Δ-5-desaturase polypeptide and/or a Δ-9elongase polypeptide or a functionally equivalent part thereof. Thesecond part of the fusion protein can be, for example, anotherpolypeptide having enzymatic activity or an antigenic polypeptidesequence by means of which it is possible to demonstrate Δ-8- and/orΔ-5-desaturase or Δ-9-elongase expression (e.g. myc tag or his tag).Preferably, however, this is a regulatory protein sequence, such as byway of example a signal sequence for the endoplasmic reticulum (=ER)which directs the Δ-8- and/or Δ-5-desaturase protein and/or theΔ-9-elongase protein to the desired point of action, or regulatorysequences which influence the expression of the nucleic acid sequenceaccording to the invention, such as promoters or terminators. In anotherpreferred embodiment the second part of the fusion protein is aplastidial targeting sequence as described by Napier J. A. [Targeting offoreign proteins to the chloroplast, Methods Mol. Biol., 49, 1995:369-376]. A preferred used vector comprising said plastidial targetingsequence is disclosed by Colin Lazarus [Guerineau F., Woolston S.,Brooks L., Mullineaux P. “An expression cassette for targeting foreignproteins into chloroplast; Nucleic. Acids Res., Dec. 9, 16 (23), 1988:11380].

Advantageously, the Δ-8-desaturase and Δ-9-elongase and/or theΔ-5-desaturase genes in the method according to the invention may becombined with other genes for fatty acid biosynthesis. Examples of suchgenes are the acyl transferases, other desaturases or elongases such asΔ-4-, Δ-5- or Δ-6-desaturases or ω-3- and/or (specific desaturases suchas Δ-12 (for C₁₈ fatty acids), Δ-15 (for C₁₈ fatty acids) or Δ-19 (forC₂₂ fatty acids) and/or such as Δ-5- or Δ-6-elongases. For in vivo andespecially in vitro synthesis combination with e.g. NADH cytochrome B5reductases which can take up or release reduction equivalents isadvantageous.

By the amino acid sequences according to the invention is meant proteinswhich contain an amino acid sequence depicted in the sequences SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10 or asequence obtainable therefrom by substitution, inversion, insertion ordeletion of one or more amino acid groups (such sequences arederivatives of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8and/or SEQ ID NO: 10), whereas the enzymatic activities of the proteinsdepicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 andSEQ ID NO: 10 being retained or not substantially reduced, that is theystill possess the same enzymatic specificity. By “not substantiallyreduced” or “the same enzymatic activity” is meant all enzymes whichstill exhibit at least 10%, preferably 20%, particularly preferably 30%,of the enzymatic activity of the initial enzyme obtained from the wildtype source organism such as organisms of the genus Physcomitrella,Ceratodon, Borago, Thraustochytrium, Schizochytrium, Phytophtora,Mortierella, Caenorhabditis, Aleuriba, Muscariodides, Isochrysis,Phaeodactylum, Crypthecodinium or Euglenia preferred source organismsare organisms such as the species Euglenia gracilis, Isochrysis galbana,Phaeodactylum tricomutum, Caenorhabditis elegans, Thraustochytrium,Phytophtora infestans, Ceratodon purpureus, Isochrysis galbana,Aleuritia farinosa, Muscariodides vialii, Mortierella alpina, Boragoofficinalis or Physcomitrella patens. For the estimation of an enzymaticactivity which is “not substantially reduced” or which has the “sameenzymatic activity” the enzymatic activity of the derived sequences aredetermined and compared with the wild type enzyme activities. In doingthis, for example, certain amino acids may be replaced by others havingsimilar physiochemical properties (space filling, basicity,hydrophobicity, etc.). For example, arginine residues are exchanged forlysine residues, valine residues for isoleucine residues or asparticacid residues for glutamic acid residues. However, one or more aminoacids may also be swapped in sequence, added or removed, or a pluralityof these measures may be combined with one another.

By derivatives is also meant functional equivalents which in particularalso contain natural or artificial mutations of an originally isolatedsequence encoding Δ-8-desaturase, a Δ-9-elongase and/or a Δ-5-desaturasewhich continue to exhibit the desired function, that is the enzymaticactivity and substrate selectivity thereof is not substantially reduced.Mutations comprise substitutions, additions, deletions, exchanges orinsertions of one or more nucleotide residues. Thus, for example, thepresent invention also encompasses those nucleotide sequences which areobtained by modification of the Δ-8-desaturase nucleotide sequence, theΔ-5-desaturase nucleotide sequence and/or the Δ-9-elongase nucleotidesequence used in the inventive processes. The aim of such a modificationmay be, e.g., to further bound the encoding sequence contained thereinor also, e.g., to insert further restriction enzyme interfaces.

Functional equivalents also include those variants whose function bycomparison as described above with the initial gene or gene fragment isweakened (=not substantially reduced) or reinforced (=enzyme activityhigher than the activity of the initial enzyme, that is activity ishigher than 100%, preferably higher than 110%, particularly preferablyhigher than 130%).

At the same time the nucleic acid sequence may, for example,advantageously be a DNA or cDNA sequence. Suitable encoding sequencesfor insertion into an expression cassette according to the inventioninclude by way of example those which encode a Δ-8-desaturase, aΔ-5-desaturase and/or a Δ-9-elongase with the sequences described aboveand lend the host the ability to overproduce fatty acids, oils or lipidshaving double bonds in the Δ-8-position and Δ-5-position, it beingadvantageous when at the same time fatty acids having at least fourdouble bonds are produced. These sequences may be of homologous orheterologous origin.

By the expression cassette (=nucleic acid construct or fragment or geneconstruct) according to the invention is meant the sequences specifiedin SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and/or SEQ IDNO: 9 which result from the genetic code and/or derivatives thereofwhich are functionally linked with one or more regulation signalsadvantageously to increase the gene expression and which control theexpression of the encoding sequence in the host cell. These regulatorysequences should allow the selective expression of the genes and theprotein expression. Depending on the host organism this may mean, forexample, that the gene is expressed and/or overexpressed only afterinduction or that d is expressed and/or overexpressed immediately.Examples of these regulatory sequences are sequences to which inductorsor repressors bind and in this way regulate the expression of thenucleic acid. In addition to these new regulation sequences or insteadof these sequences the natural regulation of these sequences ahead ofthe actual structural genes may still be present and optionally havebeen genetically modified so that natural regulation was switched offand the expression of the genes increased. However, the gene constructcan also be built up more simply, that is no additional regulationsignals have been inserted ahead of the nucleic acid sequence orderivatives thereof and the natural promoter with its regulation has notbeen removed. Instead of this the natural regulation sequence wasmutated in such a way that no further regulation ensues and/or the geneexpression is heightened. These modified promoters in the form of partsequences (=promoter containing parts of the nucleic acid sequencesaccording to the invention) can also be brought on their own ahead ofthe natural gene to increase the activity. In addition, the geneconstruct may advantageously also contain one or more so-called enhancersequences functionally linked to the promoter which allow enhancedexpression of the nucleic acid sequence. At the 3′ end of the DNAsequences additional advantageous sequences may also be inserted, suchas further regulatory elements or terminators. The Δ-8- and/orΔ-5-desaturase gene and/or the Δ-9-elongase gene may be present in oneor more copies in the expression cassette (=gene construct).

As described above, the regulatory sequences or factors can preferablypositively influence and so increase the gene expression of theintroduced genes. Thus, reinforcement of the regulatory elementsadvantageously on the transcription level may be effected by usingpowerful transcription signals such as promoters and/or enhancers.However, in addition reinforcement of translation is also possible, forexample by improving the stability of the mRNA.

Suitable promoters in the expression cassette are in principle allpromoters which can control the expression of foreign genes in organismssuch as microorganisms like protozoa such as ciliates, algae such asgreen, brown, red or blue algae such as Euglenia, bacteria such asgram-positive or gram-negative bacteria, yeasts such as Saccharomyces,Pichia or Schizosaccharomyces or fungi such as Mortierella,Thraustochytrium or Schizochytium or plants such as Aleuritia,advantageously in plants or fungi. Use is preferably made in particularof plant promoters or promoters derived from a plant virus. Advantageousregulation sequences for the method according to the invention are foundfor example in promoters such as cos, tac, trp, tet, trp-tet, Ipp, Iac,Ipp-Iac, Iacl^(q−,) T7, T5, T3, gal, trc, ara, SP6, λ-P_(R) or inλ-P_(L) promoters which are employed advantageously in gram-negativebacteria. Other advantageous regulation sequences are found, forexample, in the gram-positive promoters amy and SPO2, in the yeast orfungal promoters ADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or inthe plant promoters CaMV/35S [Franck et al., Cell 21(1980) 285-294],SSU, OCS, lib4, STLS1, B33, nos (=Nopalin Synthase Promoter) or in theubiquintin or phaseolin promoter. The expression cassette may alsocontain a chemically inducible promoter by means of which the expressionof the exogenous Δ8- and/or Δ-5-desaturase gene and/or the Δ-9-elongasegene in the organisms can be controlled advantageously in the plants ata particular time. Advantageous plant promoters of this type are by wayof example the PRP1 promoter [Ward et al., Plant. Mol. Biol. 22(1993),361-366], a promoter inducible by benzenesulfonamide (BP 388 186), apromoter inducible by tetracydine [Gatz et al., (1992) Plant J.2,397-404], a promoter inducible by salicylic acid (WO 95/19443), apromoter inducible by abscissa acid (EP 335 528) and a promoterinducible by ethanol or cyclohexanone (WO93/21334). Other examples ofplant promoters which can advantageously be used are the promoter ofcytosolic FBPase from potato, the ST-LSI promoter from potato (Stockhauset al., EMBO J. 8 (1989) 2445245), the promoter of phosphoribosylpyrophosphate amido transferase from Glycine max (see also gene bankaccession number U87999) or a no diene-specific promoter as described inEP 249 676. Particularly advantageous are those plant promoters whichensure expression in tissues or plant parts/organs in which fatty acidbiosynthesis or the precursor stages thereof occurs, as in endosperm orin the developing embryo for example. Particularly noteworthy areadvantageous promoters which ensure seed-specific expression such as byway of example the USP promoter or derivatives thereof, the LEB4promoter, the phaseolin promoter or the napin promoter. The particularlyadvantageous USP promoter cited according to the invention or itsderivatives mediate very early gene expression in seed development[Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67]. Otheradvantageous seed-specific promoters which may be used formonocotylodonous or dicotylodonous plants are the promoters suitable fordicotylodons such as napin gene promoters, likewise cited by way ofexample, from oilseed rape (U.S. Pat. No. 5,608,152), the oleosinpromoter from Arabidopsis (WO 98/45461), the phaseolin promoter fromPhaseolus vulgaris (U.S. Pat. No. 5,504,200), the Bce4 promoter fromBrassica (WO 91/13980) or the leguminous B4 promoter (LeB4, Baeumlein etal., Plant J., 2, 2, 1992: 233-239) or promoters suitable formonocotylodons such as the promoters of the Ipt2 or Ipt1 gene in barley(WO 95/15389 and WO 95/23230) or the promoters of the barley hordeinegene, the rice glutelin gene, the rice oryzin gene, the rice prolamingene, the wheat gliadin gene, the white glutelin gene, the corn zeingene, the oats glutelin gene, the sorghum kasirin gene or the ryesecalin gene which are described in WO99/16890.

Furthermore, particularly preferred are those promoters which ensure theexpression in tissues or plant parts in which, for example, thebiosynthesis of fatty acids, oils and lipids or the precursor stagesthereof takes place. Particularly noteworthy are promoters which ensurea seed-specific expression. Noteworthy are the promoter of the napingene from oilseed rape (U.S. Pat. No. 5,608,152), the USP promoter fromVicia faba (USP=unknown seed protein, Baeumlein et al., Mol Gen Genet,1991, 225 (3): 459-67), the promoter of the oleosin gene fromArabidopsis (WO98/45461), the phaseolin promoter (U.S. Pat. No.5,504,200) or the promoter of the legumin B4 gene (LeB4; Baeumlein etal., 1992, Plant Journal, 2 (2): 233-9). Other promoters to be mentionedare that of the Ipt2 or Ipt1 gene from barley (WO95/15389 andWO95/23230) which mediate seed-specific expression in monocotyledonousplants. Other advantageous seed specific promoters are promoters such asthe promoters from rice, corn or wheat disclosed in WO 99/16890 orAmy32b, Amy6-6 or aleurain (U.S. Pat. No. 5,677,474), Bce4 (rape, U.S.Pat. No. 5,530,149), glycine (soy bean, EP 571 741), phosphoenol pyruvatcarboxylase (soy bean, JP 06/62870), ADR12-2 (soy bean, WO 98/08962),isocitratlyase (rape, U.S. Pat. No. 5,689,040) or β-amylase (barley, EP781 849).

As described above, the expression construct (=gene construct, nucleicacid construct) may contain yet other genes which are to be introducedinto the organisms. These genes can be subject to separate regulation orbe subject to the same regulation region as the Δ-8- and/orΔ-5-desaturase gene and/or the Δ-9-elongase gene. These genes are by wayof example other biosynthesis genes, advantageously for fatty acidbiosynthesis, which allow increased synthesis. Examples which may bementioned are the genes for Δ-15-, Δ-12-, Δ-9-, Δ-5-, Δ-4-desaturase,α-ketoacyl reductases, α-ketoacyl synthases, elongases or the varioushydroxylases and acy-ACP thioesterases. The desaturase genes areadvantageously used in the nucleic acid construct.

In principle all natural promoters with their regulation sequences canbe used like those named above for the expression cassette according tothe invention and the method according to the invention. Over and abovethis, synthetic promoters may also advantageously be used.

In the preparation of an expression cassette various DNA fragments canbe manipulated in order to obtain a nucleotide sequence which usefullyreads in the correct direction and is equipped with a correct readingraster. To connect the DNA fragments (=nucleic acids according to theinvention) to one another adaptors or linkers may be attached to thefragments.

The promoter and the terminator regions can usefully be provided in thetranscription direction with a linker or polylinker containing one ormore restriction points for the insertion of this sequence. Generally,the linker has 1 to 10, mostly 1 to 8, preferably 2 to 6, restrictionpoints. In general the size of the linker inside the regulatory regionis less than 100 bp, frequently less than 60 bp, but at least 5 bp. Thepromoter may be both native or homologous as well as foreign orheterologous to the host organism, for example to the host plant. In the5′-3′ transcription direction the expression cassette contains thepromoter, a DNA sequence which encodes a Δ-8-desaturase gene, aΔ-5-desaturase gene and/or a Δ-9-elongase gene and a region fortranscription termination. Different termination regions can beexchanged for one another in any desired fashion.

Furthermore, manipulations which provide suitable restriction interfacesor which remove excess DNA or restriction interfaces can be employed.Where insertions, deletions or substitutions, such as transitions andtransversions, come into consideration, in vitro mutagenesis, primerrepair, restriction or ligation may be used. In suitable manipulationssuch as restriction, cheving back or filling of overhangs for blunt endscomplementary ends of the fragments can be provided for the ligation.

For an advantageous high expression the attachment of the specific ERretention signal SEKDEL inter alia can be of importance (Schouten, A etal., Plant Mol. Biol. 30 (1996), 781-792). In this way the averageexpression level is tripled or even quadrupled. Other retention signalswhich occur naturally in plant and animal proteins located in the ER mayalso be employed for the construction of the cassette. In anotherpreferred embodiment a plastidial targeting sequence is used asdescribed by Napier J. A. [Targeting of foreign proteins to thechloroplast, Methods Mol. Biol., 49, 1995: 369-376]. A preferred usedvector comprising said plastidial targeting sequence is disclosed byColin Lazarus [Guerineau F., Woolston S., Brooks L, Mullineaux P. “Anexpression cassette for targeting foreign proteins into chloroplast;Nucleic. Acids Res., Dec. 9, 16 (23), 1988: 11380].

Preferred polyadenylation signals are plant polyadenylation signals,preferably those which substantially correspond to T-DNA polyadenylationsignals from Agrobacterium tumefaciens, in particular gene 3 of theT-DNA (octopin synthase) of the Ti plasmid pTiACH5 (Gielen et al., EMBOJ. 3 (1984), 835 et seq.) or corresponding functional equivalents.

An expression cassette is produced by fusion of a suitable promoter witha suitable Δ-8- and/or Δ-5-desaturase DNA sequence and/or a suitableΔ-9-elongase DNA sequence together with a polyadenylation signal bycommon recombination and cloning techniques as described, for example,in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1989) as well as in T. J. Silhavy, M. L. Berman and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1984) and in Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Greene Publishing Assoc. andWiley-Interscience (1987).

In the preparation of an expression cassette various DNA fragments canbe manipulated to produce a nucleotide sequence which usefully reads inthe correct direction and is equipped with a correct reading raster.Adapters or linkers can be attached to the fragments for joining the DNAfragments.

The promoter and the terminator regions can usefully be provided in thetranscription direction with a linker or polylinker containing one ormore restriction points for the insertion of this sequence. Generally,the linker has 1 to 10, mostly 1 to 8, preferably 2 to 6, restrictionpoints. In general the size of the linker inside the regulatory regionis less than 100 bp, frequently less than 60 bp, but at least 5 bp. Thepromoter may be both native or homologous as well as foreign orheterologous to the host organism, for example to the host plant In the5′-3′ transcription direction the expression cassette contains thepromoter, a DNA sequence which either encodes a Δ-8- and/orΔ-5-desaturase gene and/or a Δ-9-elongase gene and a region fortranscription termination. Different termination regions can beexchanged for one another in any desired fashion.

In the preparation of an expression cassette various DNA fragments canbe manipulated to produce a nucleotide sequence which usefully reads inthe correct direction and is equipped with a correct reading raster.Adapters or linkers can be attached to the fragments for joining the DNAfragments.

The DNA sequences encoding the nucleic acid sequences used in theinventive processes such as the Δ-8-desaturase from Euglenia gracilis,the Δ-9-elongase from Isochrysis galbana and/or the Δ-5-desaturase forexample from Caenorhabditis elegans, Mortierella alpina, Borageofficinalis or Physcomitrella patens contain all the sequencecharacteristics needed to achieve correct localization of the site offatty acid, lipid or oil biosynthesis. Accordingly, no further targetingsequences are needed per se. However, such a localization may bedesirable and advantageous and hence artificially modified or reinforcedso that such fusion constructs are also a preferred advantageousembodiment of the invention.

Particularly preferred are sequences which ensure targeting in plastids.Under certain circumstances targeting into other compartments (reportedin: Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423) may also bedesirable, e.g. into vacuoles, the mitochondrium, the endoplasmicreticulum (ER), peroxisomes, lipid structures or due to lack ofcorresponding operative sequences retention in the compartment oforigin, the cytosol.

Advantageously, the nucleic acid sequences according to the invention orthe gene construct together with at least one reporter gene are clonedinto an expression cassette which is introduced into the organism via avector or directly into the genome. This reporter gene should allow easydetection via a growth, fluorescence, chemical, bioluminescence orresistance assay or via a photometric measurement. Examples of reportergenes which may be mentioned are antibiotic- or herbicide-resistancegenes, hydrolase genes, fluorescence protein genes, bioluminescencegenes, sugar or nucleotide metabolic genes or biosynthesis genes such asthe Ura3 gene, the IIv2 gene, the luciferase gene, the β-galactosidasegene, the gfp gene, the 2-desoxyglucose-6-phosphate phosphatase gene,the β-glucuronidase gene, β-lactamase gene, the neomycinphosphotransferase gene, the hygromycin phosphobansferase gene or theBASTA (=gluphosinate-resistance) gene. These genes permit easymeasurement and quantificaton of the transcription activity and hence ofthe expression of the genes. In this way genome positions may beidentified which exhibit differing productivity.

In a preferred embodiment an expression cassette comprises upstream,i.e. at the 5′ end of the encoding sequence, a promoter and downstream,i.e. at the 3′ end, a polyadenylation signal and optionally otherregulatory elements which are operably linked to the interveningencoding sequence for Δ-8-desaturase, Δ-9-elongase and/or Δ-5-desaturaseDNA sequence. By an operable linkage is meant the sequential arrangementof promoter, encoding sequence, terminator and optionally otherregulatory elements in such a way that each of the regulatory elementscan fulfill its function in the expression of the encoding sequence indue manner. The sequences preferred for operable linkage are targetingsequences for ensuring subcellular localization in plastids. However,targeting sequences for ensuring subcellular localization in themitochondrium, in the endoplasmic reticulum (=ER), in the nucleus, inoil corpuscles or other compartments may also be employed as well astranslation promoters such as the 5′ lead sequence in tobacco mosaicvirus (Gallie et al., Nucl. Acids Res. 15 (1987), 8693-8711).

An expression cassette may, for example, contain a constitutive promoteror a tissue-specific promoter (preferably the USP or napin promoter) thegene to be expressed and the ER retention signal. For the ER retentionsignal the KDEL amino acid sequence (lysine, aspartic acid, glutamicacid, leucine) or the KKX amino acid sequence (lysine-lysine-X-stop,wherein X means every other known amino acid) is preferably employed.

For expression in a prokaryotic or eukaryotic host organism, for examplea microorganism such as a fungus or a plant the expression cassette isadvantageously inserted into a vector such as by way of example aplasmid, a phage or other DNA which allows optimum expression of thegenes in the host organism. Examples of suitable plasmids are: in E.coli pLG338, pACYC184, pBR series such as e.g. pBR322, pUC series suchas pUC18 or pUC19, M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236,pMBL24, pLG200, pUR290, pIN-III¹¹³-B1, λgt11 or pBdCl; in StreptomyoespIJ101, pIJ364, pIJ702 or pIJ361; in Bacillus pUB110, pC194 or pBD214;in Corynebacterium pSA77 or pAJ667; in fungi pALS1, pIL2 or pBB116;other advantageous fungal vectors are described by Romanos, M. A. et al,[(1992) “Foreign gene expression in yeast a review”, Yeast 8: 423488]and by van den Hondel, C. A. M. J. J. et al. [(1991) “Heterologous geneexpression in filamentous fungi” as well as in More Gene Manipulationsin Fungi [J. W. Bennet & L. L. Lasure, eds., pp. 396-428: AcademicPress: San Diego] and in “Gene transfer systems and vector developmentfor filamentous fungi” [van den Hondel, C. A. M. J. J. & Punt, P. J.(1991) in: Applied Molecular Genetics of Fungi, Peberdy, J. F. et al.,eds., pp. 1-28, Cambridge University Press: Cambridge]. Examples ofadvantageous yeast promoters are 2 μM, pAG-1, YEp6, YEp13 or pEMBLYe23.Examples of algal or plant promoters are pLGV23, pGHIac⁺, pBIN19,pAK2004, pVKH or pDH51 (see Schmidt, R. and Willmitzer, L., 1988). Thevectors identified above or derivatives of the vectors identified aboveare a small selection of the possible plasmids. Further plasmids arewell known to those skilled in the art and may be found, for example, inthe book Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier,Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). Suitable plantvectors are described inter alia in “Methods in Plant Molecular Biologyand Biotechnology” (CRC Press), Ch. 6/7, pp. 71-119. Advantageousvectors are known as shuttle vectors or binary vectors which replicatein E. coli and Agrobacterium.

By vectors is meant with the exception of plasmids all other vectorsknown to those skilled in the art such as by way of example phages,viruses such as SV40, CMV, baculovirus, adenovirus, transposons, ISelements, phasmids, phagemids, cosmids, linear or circular DNA. Thesevectors can be replicated autonomously in the host organism or bechromosomally replicated, chromosomal replication being preferred.

In a further embodiment of the vector the expression cassette accordingto the invention may also advantageously be introduced into theorganisms in the form of a linear DNA and be integrated into the genomeof the host organism by way of heterologous or homologous recombination.This linear DNA may be composed of a linearized plasmid or only of theexpression cassette as vector or the nucleic acid sequences according tothe invention.

In a further advantageous embodiment the nucleic acid sequence accordingto the invention can also be introduced into an organism on its own.

If in addition to the nucleic acid sequence according to the inventionfurther genes are to be introduced into the organism, all together witha reporter gene in a single vector or each single gene with a reportergene in a vector in each case can be introduced into the organism,whereby the different vectors can be introduced simultaneously orsuccessively.

The vector advantageously contains at least one copy of the nucleic acidsequences according to the invention and/or the expression cassette(=gene construct) according to the invention.

By way of example the plant expression cassette can be installed in thepRT transformation vector ((a) Toepfer et al., 1993, Methods Enzymol.,217: 66-78; (b) Toepfer et al. 1987, Nucl. Acids. Res. 15: 5890 ff.).

Alternatively, a recombinant vector (=expression vector) can also betranscribed and translated in vitro, e.g. by using the T7 promoter andthe T7 RNA polymerase.

Expression vectors employed in prokaryotes frequently make use ofinducible systems with and without fusion proteins or fusionoligopeptides, wherein these fusions can ensue in both N-terminal andC-terminal manner or in other useful domains of a protein. Such fusionvectors usually have the following purposes: i.) to increase the RNAexpression rate; ii.) to increase the achievable protein synthesis rate;iii.) to increase the solubility of the protein; iv.) or to simplifypurification by means of a binding sequence usable for affinitychromatography. Proteolytic cleavage points are also frequentlyintroduced via fusion proteins which allows cleavage of a portion of thefusion protein and purification. Such recognition sequences forproteases are recognized, e.g. factor Xa, thrombin and enterokinase.

Typical advantageous fusion and expression vectors are PGEX [PharmaciaBiotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67: 31-40],pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which contains glutathione S-transferase (GST),maltose binding protein or protein A.

Other examples of E. coli expression vectors are pTrc [Amann et al.,(1988) Gene 69:301-315] and pET vectors [Studier et al., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) 60-89; Stratagene, Amsterdam, The Netherlands].

Other advantageous vectors for use in yeast are pYepSec1 (Baldari, etal., (1987) Embo J. 6:229-234), pMFa (Kudian and Herskowitz, (1982) Cell30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYESderivatives (Invitrogen Corporation, San Diego, Calif.). Vectors for usein filamentous fungi are described in: van den Hondel, C. A. M. J. J. &Punt, P. J. (1991) “Gene transfer systems and vector development forfilamentous fungi”, in: Applied Molecular Genetics of Fungi, J. F.Peberdy, et al., eds., pp. 1-28, Cambridge University Press: Cambridge.

Alternatively, insect cell expression vectors can also be advantageouslyutilized, e.g. for expression in Sf 9 cells. These are e.g. the vectorsof the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) andthe pVL series (Lucklow and Summers (1989) Virology 170:31-39).

Furthermore, plant cells or algal cells can advantageously be used forgene expression. Examples of plant expression vectors may be found inBecker, D., et al. (1992) “New plant binary vectors with selectablemarkers located proximal to the left border”, Plant Mol. Biol. 20:1195-1197 or in Bevan, M. W. (1984) “Binary Agrobacterium vectors forplant transformation”, Nucl. Acid. Res. 12: 8711-8721.

Furthermore, the nucleic acid sequences may also be expressed inmammalian cells, advantageously in nonhuman mammalian cells. Examples ofcorresponding expression vectors are pCDM8 and pMT2PC referred to in:Seed, B. (1987) Nature 329:840 or Kaufman et al. (1987) EMBO J. 6:187-195). At the same time promoters preferred for use are of viralorigin, such as by way of example promoters of polyoma, adenovirus 2,cytomegalovirus or simian virus 40. Other prokaryotic and eukaryoticexpression systems are referred to in chapters 16 and 17 of Sambrook etal., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989.

The host organism (=transgenic organism) advantageously contains atleast one copy of the nucleic acid according to the invention and/or ofthe nucleic acid construct according to the invention.

The introduction of the nucleic acids according to the invention, theexpression cassette or the vector into organisms, plants for example,can in principle be done by all of the methods known to those skilled inthe art. The introduction of the nucleic acid sequences gives rise torecombinant or transgenic organisms.

In the case of microorganisms, those skilled in the art can findappropriate methods in the textbooks by Sambrook, J. et al. (1989)Molecular cloning: A laboratory manual, Cold Spring Harbor LaboratoryPress, by F. M. Ausubel et al. (1994) Current protocols in molecularbiology, John Wiley and Sons, by D. M. Glover et. al., DNA Cloning Vol.1, (1995), IRL Press (ISBN 019-963476-9), by Kaiser et al. (1994)Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press orGuthrie et al. Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, 1994, Academic Press.

The transfer of foreign genes into the genome of a plant is calledtransformation. In doing this the methods described for thetransformation and regeneration of plants from plant tissues or plantcells are utilized for transient or stable transformation. Suitablemethods are protoplast transformation by poly(ethylene glycol) inducedDNA uptake, the “biolistic” method using the gene cannon—referred to asthe particle bombardment method, electroporation, the incubation of dryembryos in DNA solution, microinjection and gene transfer mediated byAgrobacterium. Said methods are described by way of example in B. Jeneset al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1,Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press(1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec.Biol. 42 (1991) 205-225). The nucleic acids or the construct to beexpressed is preferably cloned into a vector which is suitable fortransforming Agrobacterium tumefaciens, for example pBin19 (Bevan etal., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed by sucha vector can then be used in known manner for the transformation ofplants, in particular of crop plants such as by way of example tobaccoplants, for example by bathing bruised leaves or chopped leaves in anagrobacterial solution and then culturing them in suitable media. Thetransformation of plants by means of Agrobacterium tumefaciens isdescribed, for example, by Höfgen and Willmitzer in Nucl. Acid Res.(1988) 16, 9877 or is known inter alia from F. F. White, Vectors forGene Transfer in Higher Plants; in Transgenic Plants, Vol. 1,Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press,1993, pp. 15-38.

Agrobacteria transformed by an expression vector according to theinvention may likewise be used in known manner for the transformation ofplants such as test plants like Arabidopsis or crop plants such ascereal crops, corn, oats, rye, barley, wheat, soybean, rice, cotton,sugar beet, canola, sunflower, flax, hemp, potatoes, tobacco, tomatoes,carrots, paprika, oilseed rape, tapioca, cassava, arrowroot, tagetes,alfalfa, lettuce and the various tree, nut and vine species, inparticular of oil-containing crop plants such as soybean, peanut, castoroil plant, sunflower, corn, cotton, flax, oilseed rape, coconut, oilpalm, safflower (Cartharus tinctorius) or cocoa bean, e.g. by bathingbruised leaves or chopped leaves in an agrobacterial solution and thenculturing them in suitable media. For the production of PUFAs, forexample stearidonic acid, eicosapentaenoic acid and docosahexaenoicacid, borage, linseed, sunflower, safflower or Primulaceae areadvantageously suitable. Other suitable organisms for the production offor example γ-linoleic acid, dihomo-γ-linoleic acid or arachidonic acidare for example linseed, sunflower or safflower.

The genetically modified plant cells may be regenerated by all of themethods known to those skilled in the art. Appropriate methods can befound in the publications referred to above by S. D. Kung and R. Wu,Potrykus or Höfgen and Wilimitzer.

Accordingly, a further aspect of the invention relates to transgenicorganisms transformed by at least one nucleic acid sequence, expressioncassette or vector according to the invention as well as cells, cellcultures, tissue, parts—such as, for example, leaves, roots, etc. in thecase of plant organisms—or reproductive material derived from suchorganisms. The terms “host organism”, “host cell”, “recombinant (host)organism” and “transgenic (host) cell” are used here interchangeably. Ofcourse these terms relate not only to the particular host organism orthe particular target cell but also to the descendants or potentialdescendants of these organisms or cells. Since, due to mutation orenvironmental effects certain modifications may arise in successivegenerations, these descendants need not necessarily be identical withthe parental cell but nevertheless are still encompassed by the term asused here.

For the purposes of the invention “transgenic” or “recombinant” meanswith regard for example to a nucleic acid sequence, an expressioncassette (=gene construct, nucleic acid construct) or a vectorcontaining the nucleic acid sequence according to the invention or anorganism transformed by the nucleic acid sequences, expression cassetteor vector according to the invention all those constructions produced bygenetic engineering methods in which either

-   a) the nucleic acid sequence depicted in SEQ ID NO: 1, SEQ ID NO: 3,    SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or its derivatives or parts    thereof or-   b) a genetic control sequence functionally linked to the nucleic    acid sequence described under (a), for example a 3′- and/or    5′-genetic control sequence such as a promoter or terminator, or-   c) (a) and (b)    are not found in their natural, genetic environment or have been    modified by genetic engineering methods, wherein the modification    may by way of example be a substitution, addition, deletion,    inversion or insertion of one or more nucleotide residues. Natural    genetic environment means the natural genomic or chromosomal locus    in the organism of origin or inside the host organism or presence in    a genomic library. In the case of a genomic library the natural    genetic environment of the nucleic acid sequence is preferably    retained at least in part. The environment borders the nucleic acid    sequence at least on one side and has a sequence length of at least    50 bp, preferably at least 500 bp, particularly preferably at least    1,000 bp, most particularly preferably at least 5,000 bp. A    naturally occurring expression cassette—for example the naturally    occurring combination of the natural promoter of the nucleic acid    sequence according to the invention with the corresponding    Δ-8-desaturase, Δ-9-elongase and/or Δ-5-desaturase gene—turns into a    transgenic expression cassette when the latter is modified by    unnatural, synthetic (“artificial”) methods such as by way of    example a mutagenation. Appropriate methods are described by way of    example in U.S. Pat. No. 5,565,350 or WO 00/15815.

Suitable organisms or host organisms for the nucleic acid, expressioncassette or vector according to the invention are advantageously inprinciple all organisms which are able to synthesize fatty acids,especially unsaturated fatty acids or are suitable for the expression ofrecombinant genes as described above. Further examples which may bementioned are plants such as Arabidopsis, Asteraceae such as Calendulaor crop plants such as soybean, peanut, castor oil plant, sunflower,corn, cotton, flax, oilseed rape, coconut, oil palm, safflower(Carthamus tinctorius) or cocoa bean, microorganisms such as fungi, forexample the genus Mortierella, Saprolegnia or Pythium, bacteria such asthe genus Escherichia, yeasts such as the genus Saccharomyces,cyanobacteria, ciliates, algae or protozoa such as dinoflagellates likeCrypthecodinium. Preference is given to organisms which can naturallysynthesize oils in relatively large quantities such as fungi likeMortierella alpina, Pythium insidiosum or plants such as soybean,oilseed rape, coconut, oil palm, safflower, flax, castor oil plant,Calendula, peanut, cocoa bean or sunflower, or yeasts such asSaccharomyces cerevisiae and particular preference is given to soybean,flax, oilseed rape, sunflower, Calendula, Mortierella or Saccharomycescerevisiae. In principle, apart from the transgenic organisms identifiedabove, transgenic animals, advantageously nonhuman animals, aresuitable, for example C. elegans.

Further useful host cells are identified in: Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990).

Usable expression strains, e.g. those exhibiting a relatively lowprotease activity, are described in: Gottesman, S., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) 119-128.

A further object of the invention relates to the use of an expressioncassette containing DNA sequences encoding a Δ-8-desaturase, aΔ-9-elongase and/or a Δ-5-desaturase gene or DNA sequences hybridizingthere with for the transformation of plant cells, tissues or parts ofplants. The aim of use is to increase the content of fatty acids, oilsor lipids having an increased content of double bonds.

In doing so, depending on the choice of promoter, the Δ-8-desaturase, aΔ-9-elongase and/or a Δ-5-desaturase gene can be expressed specificallyin the leaves, in the seeds, the nodules, in roots, in the stem or otherparts of the plant Those transgenic plants overproducing fatty acids,oils or lipids having at least three double bonds in the fatty acidmolecule, the reproductive material thereof, together with the plantcells, tissues or parts thereof are a further object of the presentinvention.

The expression cassette or the nucleic acid sequences according to theinvention containing a Δ-8-desaturase, a Δ-9-elongase and/or aΔ-5-desaturase gene sequence can, moreover, also be employed for thetransformation of the organisms identified by way of example above suchas bacteria, cyanobacteria, yeasts, filamentous fungi, ciliates andalgae with the objective of increasing the content of fatty acids, oilsor lipids possessing at least three double bonds.

Within the framework of the present invention, increasing the content offatty acids, oils or lipids possessing at least three double bondsmeans, for example, the artificially acquired trait of increasedbiosynthetic performance due to functional overexpression of theΔ-8-desaturase, Δ-9-elongase and/or Δ-5-desaturase gene in the organismsaccording to the invention, advantageously in the transgenic plantsaccording to the invention, by comparison with the nongeneticallymodified initial plants at least for the duration of at least one plantgeneration.

The preferred locus of biosynthesis, of fatty acids, oils or lipids forexample, is generally the seed or cell layers of the seed so that aseed-specific expression of the Δ-8-desaturase, Δ-9-elongase and/orΔ-5-desaturase gene is appropriate. It is, however, obvious that thebiosynthesis of fatty acids, oils or lipids need not be limited to theseed tissue but rather can also occur in tissue-specific manner in allother parts of the plant—in epidermis cells or in the nodules forexample.

A constitutive expression of the exogenous Δ-8-desaturase, Δ-9-elongaseand/or Δ-5-desaturase gene is, moreover, advantageous. On the otherhand, however, an inducible expression may also appear desirable.

The efficiency of the expression of the Δ-8-desaturase, Δ-9-elongaseand/or Δ-5-desaturase gene can be determined, for example, in vito byshoot meristem propagation. In addition, an expression of theΔ-8-desaturase, Δ-9-elongase and/or Δ-5-desaturase gene modified innature and level and its effect on fatty acid, oil or lipid biosynthesisperformance can be tested on test plants in greenhouse trials.

An additional object of the invention comprises transgenic organismssuch as transgenic plants transformed by an expression cassettecontaining a Δ-8-desaturase, a Δ-9-elongase and/or a Δ-5-desaturase genesequence according to the invention or DNA sequences hybridizingtherewith, as well as transgenic cells, tissue, parts and reproductionmaterial of such plants. Particular preference is given in this case totransgenic crop plants such as by way of example barley, wheat, rye,oats, corn, soybean, rice, cotton, sugar beet, oilseed rape and canola,sunflower, flax, hemp, thistle, potatoes, tobacco, tomatoes, tapioca,cassava, arrowroot, alfalfa, lettuce and the various tree, nut and vinespecies.

For the purposes of the invention plants are mono- and dicotyledonousplants, mosses or algae.

A further refinement according to the invention are transgenic plants asdescribed above which contain a nucleic acid sequence according to theinvention or a expression cassette according to the invention.

Other objects of the invention are:

-   -   A method for the transformation of a plant comprising the        introduction of expression cassettes according to the invention        containing a Δ-8-desaturase, a Δ-9-elongase and/or a        Δ-5-desaturase gene sequence derived from algae such as Euglenia        or Isochrysis, fungi such as Mortierella or mosses such as        Physcomitrella or DNA′ sequences hybridizing therewith into a        plant cell, into callus tissue, an entire plant or protoplasts        of plants.    -   A method for producing PUFAs, wherein the method comprises the        growing of a transgenic organism comprising a nucleic acid as        des herein or a vector encoding a Δ-8-desaturase, a Δ-9-elongase        and/or a Δ-5-desaturase which specifically synthesize poly        unsaturated fatty acids with at least three double bonds in the        fatty acid molecule    -   Use of a Δ-8-desaturase, a Δ-9-elongase and/or a Δ-5-desaturase        DNA gene sequence or DNA sequences hybridizing therewith for the        production of plants having an increased content of fatty acids,        oils or lipids having at least three double bonds due to the        expression of said Δ-8-desaturase, Δ-9-elongase and/or        Δ-5-desaturase DNA sequence in plants.    -   Proteins containing the amino acid sequences depicted in SEQ ID        NO: 2, SEQ ID NO: 8 or its derivatives.    -   Use of said proteins having the sequences SEQ ID NO: 2 or SEQ ID        NO: 8 for producing unsaturated fatty acids.

A further object according to the invention is a method for producingunsaturated fatty acids comprising: introducing at least one saidnucleic acid sequence described herein or at least one nucleic acidconstruct or vector containing said nucleic acid sequence into apreferably oil-producing organism such as a plant or a fungi; growingsaid organism; isolating oil contained in said organism; and liberatingthe fatty acids present in said oil. These unsaturated fatty acidsadvantageously contain at least three double bonds in the fatty acidmolecule. The fatty acids may be liberated from the oils or lipids, forexample by basic hydrolysis, e.g. using NaOH or KOH or by acidhydrolysis preferably in the presence of an alcohol such as methanol orethanol. Said fatty acid liberation leads to free fatty acids or to thecorresponding alkyl esters of the fatty acids. In principle an enzymatichydrolysis for example with a lipase as enzyme is also possible.Starting from said free fatty acids or fatty acid alkyl esters mono-,di- and/or triglycerides can be synthesized either chemically orenzymatically. In another preferred embodiment of the inventive processthe alkyl ester of the fatty acids are produced from the oils and lipidsby transesterification with an enzyme of with conventional chemistry. Apreferred method is the production of the alkyl ester in the presence ofalcohalates of the corresponding lower alcohols (C1 to C10 alcohols suchas methanol, ethanol, propanol, butanol, hexanol etc.) such asmethanolate or ethanolate. Therefore as the skilled worker knows thealcohol in the presence of a catalytic amount of a base such as NaOH orKOH is added to the oils or lipids.

A method for producing triglycerides having an increased content ofunsaturated fatty acids comprising: introducing at least one saidnucleic acid sequence according to the invention or at least oneexpression cassette according to the invention into an oilproducingorganism; growing said organism; and isolating oil contained in saidorganism; is also numbered among the objects of the invention.

A further object according to the invention is a method for producingtriglycerides having an increased content of unsaturated fatty acids byincubating triglycerides containing saturated or unsaturated orsaturated and unsaturated fatty acids with at least one of the proteinsencoded by the sequences SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8 or SEQ ID NO: 10. The method is advantageously carried out inthe presence of compounds which can take up or release reductionequivalents. The fatty acids can then be liberated from thetriglycerides.

A further object according to the invention of said method for producingtriglycerides having an increased content of unsaturated fatty acidsadvantageously having an increased content of unsaturated fatty acids isa method wherein the fatty acids are liberated from the triglycerideswith the aid of basic hydrolysis known to those skilled in the art or bymeans of an enzyme such as a lipase.

The methods specified above advantageously allow the synthesis of fattyacids or triglycerides having an increased content of fatty acidscontaining at least three double bonds in the fatty acid molecule.

The methods identified above advantageously allow the synthesis of fattyacids or triglycerides having an increased content of fatty acidscontaining at least three double bonds, wherein the substrate used forthe reaction of the Δ-8-desaturase, Δ-9-elongase and/or Δ-5-desaturaseis preferably—linoleic acid (C_(20:2) ^(Δ9,12)) acid and/or α-linolenicacid (C_(18:2) ^(Δ9,12,15)). In this way the method identified aboveadvantageously allows in particular the synthesis of fatty acids derivedfrom linoleic acid (C_(20:2) ^(Δ9,12)), α-linolenic acid (C_(18:2)^(Δ9,12,15)) γ-linoleic acid (C_(18:3) ^(Δ6,9,12)), stearidonic acid(C_(18:4) ^(Δ6,9,12,15)), dihomo-γ-linoleic acid (C_(20:3) ^(Δ8,11,14))or such as by way of example eicosapentaenoic acid and arachidonic acid.

Examples of organisms for the said methods as described above are plantssuch as Arabidopsis, Primulaceae, borage, barley, wheat, rye, oats,corn, soybean, rise, cotton, sugar beet, oilseed rape and canola,sunflower, flax, hemp, potatoes, tobacco, tomatoes, rape, tapioca,cassava, arrowroot, alfalfa, peanut, castor oil plant, coconut, oilpalm, safflower (Carthamus tinctorius) or cocoa bean, microorganismssuch as the fungi Mortierella, Saprolegnia or Pythium, bacteria such asthe genus Escherichia, cyanobacteria, yeasts such as the genusSaccharomyces, algae or protozoa such as dinoflagellates likeCrypthecodinium. Preference is given to organisms which can naturallysynthesize oils in relatively large quantities such as fungi likeMortierella alpina, Pythium insidiosum or plants such as soybean,oilseed rape, coconut, oil palm, safflower, castor oil plant, Calendula,peanut, cocoa bean or sunflower, or yeasts such as Saccharomycescerevisiae and particular preference is given to soybean, oilseed rape,sunflower, flax, Primulaceae, borage, Carthamus or Saccharomycescerevisiae.

Depending on the host organism, the organisms used in the methods aregrown or cultured in the manner known to those skilled in the art.Microorganisms such as fungi or algae are usually grown in a liquidmedium containing a carbon source, usually in the form of sugars, anitrogen source, usually in the form of organic nitrogen sources such asyeast extract or salts such as ammonium sulfate, trace elements such asiron, manganese or magnesium salts and optionally vitamins attemperatures of between 10° C. and 60° C., preferably between 15° C. and40° C. with exposure to gaseous oxygen. In doing so the pH of thenutrient liquid may be kept at a fixed value, that is during growth itis or is not regulated. Growth can ensue in batch mode, semibatch modeor continuously. Nutrients can be provided at the start of fermentationor be fed in semicontinuously or continuously.

After transformation plants are first of all regenerated as describedabove and then cultured or cultivated as normal.

After growth the lipids are isolated from the organisms in the usualway. For this purpose, after harvesting the organisms may first of allbe digested or used directly. The lipids are advantageously extractedusing suitable solvents such as a polar solvents like hexane or ethanol,isopropanol or mixtures such as hexane/isopropanol,phenol/chloroform/isoamyl alcohol at temperatures of between 0° C. and80° C., preferably between 20° C. and 50° C. The biomass is usuallyextracted with an excess of solvent, for example an excess of solvent tobiomass of 1:4. The solvent is then removed, for example bydistillation. Extraction can also be done using supercritical CO₂. Afterextraction the remaining biomass may be removed, for example byfiltration.

The crude oil isolated in this way can then be further purified, forexample by removing cloudiness by treatment with polar solvents such asacetone or chloroform and then filtration or centrifugation. Furtherpurification through columns is also possible.

In order to obtain the free acids from the triglycerides the latter aresaponified in the usual way.

A further object of the invention comprises unsaturated fatty acids andtriglycerides having an increased content of unsaturated fatty acidsproduced by the methods identified above and use thereof for producingfoods, animal feeds, cosmetics or pharmaceuticals. For this purpose thelatter are added in customary quantities to the foods, the animal feed,the cosmetics or pharmaceuticals.

Said unsaturated fatty acids according to the invention as well astriglycerides having an increased content of unsaturated fatty acidsproduced by the methods identified above are the result of theexpression of the nucleic acids according to the invention in thevarious host organisms. This results overall in a modification of thecomposition of the compounds in the host cell containing unsaturatedfatty acids by comparison with the original starting host cells which donot contain the nucleic acids. These modifications are more marked inhost organisms, for example plant cells, which naturally do not containthe proteins or enzymes encoded by the nucleic acids than in hostorganisms which naturally do contain the proteins or enzymes encoded bythe nucleic acids. This gives rise to host organisms containing oils,lipids, phospholipids, sphingofipids, glycoipids, triacylglycerolsand/or free fatty acids having a higher content of PUFAs with at leastthree double bonds. For the purposes of the invention, by an increasedcontent is meant that the host organisms contain at least 5%,advantageously at least 10%, preferably at least 20%, particularlypreferably at least 30%, most particularly preferably at least 40% morepolyunsaturated fatty acids by comparison with the initial organismwhich does not contain the nucleic acids according to the invention.This is particularly the case for plants which do not naturally containlonger-chain polyunsaturated C₂₀ or C₂₂ fatty acids such as EPA or ARA.Due to the expression of the nucleic adds novel lipid compositions areproduced by said means these being a further aspect of the invention.

The invention is explained in more detail by the following examples.

EXAMPLES Example 1 General Cloning Methods

The cloning methods, such as by way of example restriction cleavages,agarose gel electrophoresis, purification of DNA fragments, transfer ofnucleic acids to nitrocellulose and nylon membranes, linkage of DNAfragments, transformation of—Escherichia coli cells, culture of bacteriaand sequence analysis of recombinant DNA, were carried out as describedin Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN0-87969-309-6).

Example 2 Sequence Analysis of Recombinant DNA

Sequencing of recombinant DNA molecules was done using a laserfluorescence DNA sequencer from the ABI company by the method of Sanger(Sanger et al. (1977) Proc. Natl. Acad. Sci. USA74, 54635467). Fragmentsresulting from a polymerase chain reaction were sequenced and checked toprevent polymerase errors in the constructs to be expressed.

Example 3 Cloning of the Δ-8-Desaturase from Euglena gracilis (=SEQ IDNO: 1)

As a template for PCR amplification, cDNA from Euglena gracilis Strain Zwas used. The cDNA was synthesised from total RNA extracted fromcultures of E. gracilis strain Z. Unique primers to the initiatingmethionine and the stop codon of the Euglena Δ-8-desaturase weresynthesized as shown, including restriction sites as detailed

Primer 1: EDELTA8BamF ATGGATCCACCATGAAGTCAAAGCGCCAA (SEQ ID NO: 11)Primer 2: EDELTA8XhoR ATCTCGAGTTATAGAGCCTTCCCCGC (SEQ ID NO: 12) PCRprotocolAddition temperature: 1 min at 45° C.Denaturing temperature: 1 min at 94° C.Elongation temperature: 2 min at 72° C.Number of cycles: 30

The PCR products were separated on an agarose gel and a 1270 bp fragmentwas isolated. The PCR fragment was cloned in the pGEM-T easy vector(Promega) and the insert was then sequenced. This revealed the presenceof an open reading frame of 1266 base pairs, encoding a protein of 421amino acid residues and a stop codon. The C-terminals of the clonedΔ-8-desaturase has high homologies to the Δ-8-desaturase published byWallis and Browse (Archives of Biochem. and Biophysics, Vol. 365, No. 2,1999) which is reported to be an enzyme of 422 residues; see alsorelated sequence by these authors [GenBank AF139720/AAD45877] whichpurports to relate to the same Δ-8-desaturase but describes an openreading frame of 419 residues]. The deduced amino acid sequence theEuglena Δ-8-desaturase described in this present invention differs fromthat previously described by heterogeneity at the N-terminus. Inparticular, the first 25 amino acid residues of LARS Δ-8-desaturase is:

MKSKRQALP LTIDGTTDVS AWVNF (SEQ ID NO: 13)Whereas the sequence described by Wallis & Browse is:

MKSKRQALS PLQLMEQTYDV SAWVN (SEQ ID NO: 14)Or, alternatively

MKSKRQALSPLQLMEQTYDVVNFH (SEQ ID NO: 15)(as given in GenBank AAD45877)

Said heterogeneity present at the N-terminus of the desaturase sequenceis not resultant of the PCR amplification or primers. The distinctionsare true differences between the proteins.

Example 4 Construction of Transgenic Plants Expressing the Isochrysisgalbana Elongase Component IgASE1

The cloning of IgASE1 cDNA is described in: Qi, B., Beaudoin, F.,Fraser, T., Stobart, A. K., Napier, J. A. and Lazarus, C. M.Identification of a cDNA encoding a novel C18-Δ-9-polyunsaturated fattyacid-specific elongating activity from the docosahexaenoic acid(DHA)-producing microalga, Isochrysis galbana. FEBS Letters 510, 159-165(2002). The cDNA was released from plasmid vector pCR2.1-TOPO bydigestion with KpnI, and ligated into the KpnI site of the intermediatevector pBlueBac 4.5 (Invitrogen). Recombinant plasmids were screened forinsert orientation with EcoRI. The insert was released from a selectedplasmid with PstI plus EcoRI and ligated into binary vector plasmidpCB302-1 (Xiang et al, 1999) that had been cut with the same enzymes.This placed the IgASE1 coding region under the control of the CaMV 35Spromoter as a translational fusion with the transit peptide of the smallsubunit of Rubisco (Xiang at al., 1999), with the intention of targetingthe elongase component to chloroplasts when expressed in transgenicplants. This recombinant binary vector was designated pCB302-1ASE. Toconstruct a similar vector with expression of the elongase componenttargeted to the microsomal membrane, the IgASE1 coding region wasremoved from the intermediate vector by digestion with BamHI plus SpeI,and ligated into the corresponding sites of pCB302-3 (Xiang et al.,1999, in which the map of pCB302-3 is incorrect the CaMV 35S promoter(plus omega sequence) and nos terminator regions are reversed withrespect to MCS2). This recombinant binary vector was designatedpCB302-3ASE.

Example 5 Plant Expression of the Elongase

Binary vectors were transferred to Agrobacterium tumefaciens strainGV3101 by electroporation; transformed colonies were selected on mediumcontaining 50 μg ml⁻¹, kanamycin. Selected colonies were gown tostationary phase at 28° C., then the cells were concentrated bycentrifugation and resuspended in a dipping solution containing 5%sucrose, 0.03% Silwet-177 and 10 mM MgCl₂.

Seeds of Arabidopsis thaliana ecotype Columbia 4 were germinated onone-half-strength Murashige and Skoog medium, and seedlings weretransferred to compost in 15 cm flower pots. Plants were grown toflowering stage in a growth cabinet at 21° C., with a 23 light and 1hour dark cycle. Plant transformation was carried out by the floraldipping method of Clough and Bent (1998, Floral dip: a simplified methodfor Agrobacterium-mediated transformation of Arabidopsis thaliana. PlantJournal 16, 735-743 (1998), essentially as follows:

For each construct two pots containing 16 plants were inverted in thedipping solutions containing transformed A. tumefaciens (describedabove). The plants were then covered with a plastic bag and left at roomtemperature in the dark overnight. The bag was then removed and theplants transferred to the growth cabinet. Dipping (with fresh A.tumefaciens solutions) was repeated after 5 days and the plants wereallowed to set seed. Bulked seed from dipped plants (=T1 seed) wascollected, and approximately 10000 seed sprinkled onto compost in a seedtray, and, after stratification at 4° C. for 2 days, cultivated in thegrowth cabinet. When seedlings had reached the 2 to 4 trueleaf stagethey were sprayed with Liberty herbicide (Aventis, 0.5 gglufosinate-ammonium ml⁻¹), and spraying was repeated one week later.Twelve herbicide-resistant plants were selected and potted on for eachline (chloroplast or cytoplasm targeted elongase component), and allowedto self fertilize. Samples of T2 seed collected from these plants weregerminated on one-half-strength Murashige and Skoog medium containingLiberty (5 mg glufosinate-ammonium ml⁻¹). T3 seed collected fromindividual surviving plants was then again germinated on Liberty platesto screen for lines that had ceased segregating for herbicideresistance. Total fatty acids extracted from leaves of such lines wereanalysed and those with the greatest C20 content (CB12-4 with thechloroplast-targeted elongase component and CA1-9 with thecytoplasm-targeted elongase component) selected.

Example 6 Production of Transgenic Plants Expressing the Isochrysisgalbana Elongase Component IgASE1 and the Euglena gracilis Δ8 DesaturaseEUGD8

The Δ-8-desaturase coding region was removed from the yeast expressionvector pESC-Trp with BamHI plus XhoI, ligated into the BamHI and XhoIsites of pBlueBac 4.5 (Invitrogen) and transformed into E. coli strainTam 1. The insert was removed from a recombinant plasmid with BgI andBamHI, ligated into the BamHI site of pBECKS₁₉.6 and transformed into E.coli strain Tam1. DNA minipreparations were made of the recombinantplasmids of 6 transformant colonies; these were digested with XhoI todetermine the orientation of insertion of the desaturase coding regionin the binary vector. One recombinant plasmid with the insert in thecorrect orientation for expression from the CaMV 35S promoter wastransferred to Agrobacterium tumefaciens strain GV3101 byelectroporation and a dipping solution prepared from a transformedcolony as described above.

Arabidopsis thaliana lines CB12-4 and CA1-9 (see above) were subjectedto floral dipping as described above. Approximately 2000 T1-seed fromeach line were spread on 15 cm petri dishes containing one-half-strengthMurashige and Skoog (solid) medium supplemented with 50 μg ml⁻¹kanamycin and germinated in the growth cabinet. 12 kanamycin-resistantplants of the CA1-9 parental line and 3 plants of the CB12-4 parentalline were transferred to potting compost and further cultivated in thegrowth room. Fatty acid analysis was conducted on a lea taken from eachof the T2 plants, which were allowed to mature and set seed.

REFERENCES

-   McCormac, A. C., Eliott, M. C. and Chen, D-F.; pBECKS. A flexible    series of binary vectors for Agrobacterium-mediated plant    transformation. Molecular Biotechnology 8, 199-213 (1997).-   Xiang, C., Han, P., Lutziger, I., Wang, K. and Oliver, D. J.; A mini    binary vector series for plant transformation. Plant Molecular    Biology 40, 711-717 (1999).

Example 7 Production of Transgenic Plants Expressing the Isochrysisgalbana Elongase Component IgASE1 and the Euglena gracilis Δ8 DesaturaseEUGD8 and a Δ5 Desaturase

The Δ5 desaturase from Phaeodactylum tricornutum was cloned into thepGPTV plasmid (Becker, D. et al.; Plant Mol. Biol. 20 (1992), 1195-1197)harboring a hygromycin resistence selectable marker gene. Forseed-specific expression the USP promoter from Vicia faber was cloned5′-prime to the ATG of the Δ5 desaturase.

The binary vector was transferred to Agrobacterium tumefaciens strain GV3101 and transformed colonies were selected on medium containing 30 μgml⁻¹ hygromycin. Selected Agrobacteria were used for the transformation(flower transformation) of Arabidopsis plants carrying the T-DNAinsertions with the Δ9 elongase and the Δ5 desaturase.

Arabidopsis thaliana seedlings were germinated on Murashige and Skoogmedium containing hygromycin and resistent plants were transferred tothe greenhouse.

Seeds collected from individual plants were harvested and the totalfatty acid profile was analyzed using GC methods.

Example 8 Cloning of Expression Plasmids for Seed-Specific Expression inPlants

pBin-USP is a derivative of the plasmid pBin19. pBin-USP was producedfrom pBin19 by inserting a USP promoter as an EcoRI-BaMHI fragment intopBin19 (Bevan et al. (1980) Nucl. Acids Res. 12, 8711). Thepolyadenylation signal is that of gene 3 of the T-DNA of the Ti plasmidpTiACH5 (Gielen et al., (1984) EMBO J. 3, 835), whereby nucleotides11749-11939 were isolated as a PvuII-HindIII fragment and after additionof SphI linkers to the PvuII interface between the SpHI-HindIIIinterface of the vector were cloned. The USP promoter corresponds tonucleotides 1-684 (gene bank accession number X56240), wherein a part ofthe nonencoding region of the USP gene is contained in the promoter. Thepromoter fragment running to 684 base pairs was amplified by standardmethods by means of commercial T7 standard primer (Stratagene) and usinga synthesized primer through a PCR reaction.

Primer sequence:

5′-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-3′ (SEQID NO: 16)

The PCR fragment was cut again using EcoRI/SaII and inserted into thevector pBin19 with OCS terminator. The plasmid having the designationpBinUSP was obtained. The constructs were used for transformingArabidopsis thaliana, oilseed rape, tobacco and linseed.

Example 9 Production of Transgenic Oil Crops

Production of transgenic plants (modified in accordance with Moloney etal., 1992, Plant Cell Reports, 8:238-242)

To produce transgenic oilseed rape plants binary vectors inAgrobacterium tumefaciens C58C1:pGV2260 or Escherichia coli were used(Deblaere et al, 1984, Nucl. Acids. Res. 13, 4777-4788). Fortransforming oilseed rape plants (var. Drakkar, NPZ NordeutschePflanzenzucht, Hohenlieth, Germany) a 1:50 dilution of an overnightculture of a positively transformed agrobacteria colony inMurashige-Skoog medium (Murashige and Skoog 19862 Physiol. Plant. 15,473) containing 3% of saccharose (3MS medium) was used. Petioles orhypocotyledons of freshly germinated sterile rape plants (approx 1 cm²each) were incubated in a Petri dish with a 1:50 agrobacteria dilutionfor 5-10 minutes. This was followed by 3-day concubation in darkness at25° C. on 3MS medium containing 0.8% of Bacto-Agar. After three days,culturing was continued with 16 hours of light/8 hours of darkness andin a weekly cycle on MS medium containing 500 mg/l of Claforan (sodiumcefotaxime), 50 mg/l of kanamycin, 20 microM of benzylaminopurine (BAP)and 1.6 g/l of glucose. Growing shoots were transferred onto MS mediumcontaining 2% of saccharose, 250 mg/l of Claforan and 0.8% ofBacto-Agar. If after three weeks no roots had formed 2-indolylbutyricacid was added to the medium as a growth hormone for rooting purposes.

Regenerated shoots were obtained on 2MS medium using kanamycin andClaforan, transferred into soil after rooting and after culturing grownfor two weeks in a climate-controlled chamber, brought to blossom andafter harvesting of ripe seed investigated for Δ-8-desaturase expressionby means of lipid analyses. Ones having increased contents of doublebonds at the Δ-8-position were identified. In the stably transformedtransgenic lines functionally expressing the transgene it was found thatthere is an increased content of double bonds at the Δ-8-position bycomparison with untransformed control plants.

The same procedure was done to create plants with Δ-9-elongase and/orΔ-5-desaturase activity.

a) Transgenic Flax Plants

Transgenic flax plants may be produced, for example by the by the methodBell et al., 1999, In Vitro Cell. Dev. Biol.-Plant. 35(6):456-465, bymeans of partide bombardment. Agrobacteria-mediated transformations canbe produced, for example, as described by Mlynarova et al. (1994), PlantCell Report 13: 282-285.

Example 10 Lipid Extraction from Seed and Leave Material

Plant material (approx 200 mg) was first of all mechanically homogenizedby means of triturators in order to render it more amenable toextraction.

The disrupted cell sediment was hydrolyzed with 1 M methanorichydrochloric add and 5% dimethoxypropane for 1 h at 85° C. and thelipids were transmethylated. The resultant fatty acid methyl esters(FAMES) were extracted in hexane. The extracted FAMEs were analyzed bygas-liquid chromatograph using a capillary column (Chrompack, WCOT fusedsilica, CP wax 52 CB, 25 m, 0.32 mm) and a temperature gradient of from170° C. to 240° C. in 20 min and 5 min at 240° C. The identity of thefatty add methyl esters was confirmed by comparison with correspondingFAME standards (Sigma). The identity and the position of the double bondwas further analyzed by means of GC-MS by suitable chemicalderivatization of the FAME mixtures, e.g. to form 4,4-dimethoxyoxazolinederivatives (Christie, 1998).

FIG. 1 shows the fatty acid profile (FAMes) of leaf tissue from wildtypeArabidopsis thaliana as a control. FIG. 2 shows the fatty acid profile(FAMes) of leaf tissue from transgenic Arabidopsis expressing theIsochrysis Δ-9-elongase (see example 4). This Arabidopsis line wassubsequently retransformed with the Euglena Δ-8-desaturase. The fattyacid profile (FAMes) of said double transformed Arabidopsis line (LineIsoEIo X Eu D8 des) is given in FIG. 3.

Furthermore this double transformed Arabidopsis line (Line IsoElo×Eu D8des) was subsequently re-transformed with the Mortierella Δ5 desaturase(Mort Δ5) gene. The fatty acid profile (FAMes) of said tripletransformed Arabidopsis line (Line IsoElo×EU D8 des×Mort Δ5) is given inFIG. 4.

Example 11 GC Profiles of Arabidopsis Leaf Fatty Acid Methyl Esters fromDifferent Transgenics

FIG. 5 shows GC profiles of Arabidopsis leaf fatty acid methyl estersextracted from wild type (WT 5a), single transgenic plants expressingIsochrysis galbana Δ9 elongase gene Ig ASE1 (5b), double transgenicplant expressing the Ig ASE1 and Euglena Δ8 desaturase (EU Δ8) genes(5c) and the triple transfehic plant expressing the Ig ASE1, Eu Δ8 andthe Mortierella Δ5 desaturase (Mort Δ5) genes (5d).

Table 1 shows the fatty acid composition of Arabidopsis plants preparedfrom wild type (Wt), single transgenic plant expressing the Isochrysisgalbana IgASE1 elongase gene, double transgenic plants expressing theIgASE1 elongase gene and the Euglena Δ8 desaturase gene and tripletransgenic plants expressing the IgASE1, the Euglena Δ8 and theMortierella Δ5 desaturase gene. Analysis is of leaf tissue from rosettestage Arabidopsis plants. Each value represents the average of 2measurements.

Plant source IgASE1 + Fatty acid IgASE1 + EuΔ8 + (mol % of IgASE1 EuΔ8MortΔ5 total) Wt transgenic transgenic transgenic 16:0 19.9 19.2 14.714.2 16:1 2.8 3.3 1.8 2.3 16:3 13.1 12.2 19.9 15.4 18:0 1.7 2.4 0.8 1.518:1n-9 1.7 5.1 1.6 3.4 18:2n-6 11.2 9.0 4.2 6.6 18:3n-3 50.1 31.0 36.031.2 20:2n-6 — 7.9 0.9 3.2 20:3, — 1.5 Δ5, 11, 14 20:3n-6 — — 9.1 1.520:4n-6 — — 6.6 (ARA) 20:3n-3 — 9.9 4.0 4.8 20:4Δ5, — — — 1.6 11, 14, 1720:4n-3 — — 7.2 2.9 20:5n-3 — — — 3.3 (EPA) Total C20 — 17.8 21.2 22.2PUFAs

All transgenes are under the control of the 35S-CaMV viral promoter.Isochrysis Δ9 elongase (IgASE1) with SSU Rubisco transit sequence [T-DNABasta-r] were retrans-formed with Euglena Δ8-desaturase^(mut175+313)[T-DNA Kanamycin-r]. The double transformed line, which is homozygousfor both Basta-r and Kanamycin-r, were transformed again withMortierella Δ5 desaturase (T-DNA Hygromycin-r). The resulting tripletransformed line is homozygous for both Basta-r and Kanamycin-r, butheterozygous for Hygromycin-r.

1. A process for the production of one or more C₁₆-, C₁₈-, and/orC₂₀-polyunsaturated fatty acids in a transgenic organism comprising: a)introducing at least one nucleic acid sequence encoding a Δ-9-elongaseinto an organism, wherein said Δ-9-elongase comprises the amino acidsequence of SEQ ID NO: 4, b) introducing at least one second nucleicacid sequence encoding a Δ-8-desaturase, wherein the Δ-8-desaturasecomprises the amino acid sequence of SEQ ID NO: 2, c) introducing atleast one third nucleic acid sequence encoding a Δ-5-desaturase, whereinthe Δ-5-desaturase comprises the amino acid sequence of SEQ ID NO: 6,and d) cultivating and harvesting said organism, wherein said organismis a plant or a microorganism.
 2. The process of claim 1, wherein thenucleic acid sequence encoding a Δ-9-elongase comprises the nucleic acidsequence of SEQ ID NO:
 3. 3. The process of claim 1, wherein the nucleicacid sequence encoding a Δ-8-desaturase comprising the nucleic acidsequence of SEQ ID NO:
 1. 4. The process of claim 1, wherein the nucleicacid sequence encoding a Δ-5-desaturase comprises the nucleic acidsequence of SEQ ID NO:
 5. 5. The process of claim 1, wherein the nucleicacid sequence encoding a Δ-9-elongase comprises the nucleic acidsequence of SEQ ID NO: 3, the nucleic acid sequence encoding aΔ-8-desaturase comprising the nucleic acid sequence of SEQ ID NO: 1, andthe nucleic acid sequence encoding a Δ-5-desaturase comprises thenucleic acid sequence of SEQ ID NO:
 5. 6. The process of claim 1,wherein the plant is an oil producing plant.
 7. The process of claim 6,wherein the oil producing plant is selected from the group consisting ofrapeseed, poppy, mustard, hemp, castor bean, sesame, olive, calendula,punica, hazel nut, almond, macadamia, avocado, pumpkin, walnut, laurel,pistachio, primrose, canola, peanut, linseed, soybean, safflower,sunflower and borage.
 8. The process of claim 1, wherein thepolyunsaturated fatty acids are isolated in the form of oils, lipids offree fatty acids.
 9. The process of claim 1, wherein the polyunsaturatedfatty acids have at least two double bonds.
 10. A process for theproduction of compounds comprising one or more C₁₆-, C₁₈-, and/orC₂₀-polyunsaturated fatty acids in a transgenic organism comprising: a)introducing at least one nucleic acid sequence encoding a Δ-9-elongaseinto an organism, wherein said Δ-9-elongase comprises the amino acidsequence of SEQ ID NO: 4, b) introducing at least one second nucleicacid sequence encoding a Δ-8-desaturase, wherein the Δ-8-desaturasecomprises the amino acid sequence of SEQ ID NO: 2, c) introducing atleast one third nucleic acid sequence encoding a Δ-5-desaturase, whereinthe Δ-5-desaturase comprises the amino acid sequence of SEQ ID NO: 6,and d) cultivating and harvesting said organism, wherein said organismis a plant or a microorganism.
 11. The process of claim 10, wherein thenucleic acid sequence encoding a Δ-9-elongase comprises the nucleic acidsequence of SEQ ID NO:
 3. 12. The process of claim 10, wherein thenucleic acid sequence encoding a Δ8-desaturase comprising the nucleicacid sequence of SEQ ID NO:
 1. 13. The process of claim 10, wherein thenucleic acid sequence encoding a Δ-5-desaturase comprises the nucleicacid sequence of SEQ ID NO:
 5. 14. The process of claim 10, wherein thenucleic acid sequence encoding a Δ-9-elongase comprises the nucleic acidsequence of SEQ ID NO: 3, the nucleic acid sequence encoding aΔ-8-desaturase comprising the nucleic acid sequence of SEQ ID NO: 1, andthe nucleic acid sequence encoding a Δ-5-desaturase comprises thenucleic acid sequence of SEQ ID NO:
 5. 15. The process of claim 10,wherein the plant is an oil producing plant.
 16. The process of claim15, wherein the oil producing plant is selected from the groupconsisting of rapeseed, poppy, mustard, hemp, castor bean, sesame,olive, calendula, punica, hazel nut almond, macadamia, avocado, pumpkin,walnut, laurel, pistachio, primrose, canola, peanut, linseed, soybean,safflower, sunflower and borage.
 17. The process of claim 10, whereinthe compounds are isolated in the form of oils, lipids of free fattyacids.
 18. The process of claim 10, wherein the polyunsaturated fattyacids have at least two double bonds.
 19. A process for increasing thecontent of fatty acids, oils or lipids containing C₁₆-, C₁₅-, and/orC₂₀-polyunsaturated fatty acids in an organism comprising: a)introducing at least one nucleic acid sequence encoding a Δ-9-elongaseinto an organism, wherein said Δ-9-elongase comprises the amino acidsequence of SEQ ID NO: 4, b) introducing at least one second nucleicacid sequence encoding a Δ-8-desaturase, wherein the Δ-8-desaturasecomprises the amino acid sequence of SEQ ID NO: 2, c) introducing atleast one third nucleic acid sequence encoding a Δ-5-desaturase, whereinthe Δ-5-desaturase comprises the amino acid sequence of SEQ ID NO: 6,and d) cultivating and harvesting said organism, wherein said organismis a plant or a microorganism.
 20. The process of claim 19, wherein thenucleic acid sequence encoding a Δ-9-elongase comprises the nucleic acidsequence of SEQ ID NO:
 3. 21. The process of claim 19, wherein thenucleic acid sequence encoding a Δ-8-desaturase comprising the nucleicacid sequence of SEQ ID NO:
 1. 22. The process of claim 19, wherein thenucleic acid sequence encoding a Δ-5-desaturase comprises the nucleicacid sequence of SEQ ID NO:
 5. 23. The process of claim 19, wherein thenucleic acid sequence encoding a Δ-9-elongase comprises the nucleic acidsequence of SEQ ID NO: 3, the nucleic acid sequence encoding aΔ-5-desaturase comprising the nucleic acid sequence of SEQ ID NO: 1, andthe nucleic acid sequence encoding a Δ-5-desaturase comprises thenucleic acid sequence of SEQ ID NO:
 5. 24. The process of claim 19,wherein the plant is an oil producing plant.
 25. The process of claim24, wherein the oil producing plant is selected from the groupconsisting of rapeseed, poppy, mustard, hemp, castor bean, sesame,olive, calendula, punica, hazel nut, almond, macadamia, avocado,pumpkin, walnut, laurel, pistachio, primrose, canola, peanut, linseed,soybean, safflower, sunflower and borage.
 26. The process of claim 19,wherein the polyunsaturated fatty acids have at least two double bonds.